Stable formulations for parenteral injection of peptide drugs

ABSTRACT

Stable formulations for parenteral injection of peptide drugs and methods of using such stable formulations are provided. In particular, the present invention provides stable formulations for parenteral injection of glucagon and methods of using such glucagon formulations to treat hypoglycemia, especially severe hypoglycemia in emergency situations.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 61/451,568, filed Mar. 10, 2011, and entitled “StableFormulations for Parenteral Injection of Peptide Drugs”; of U.S.Provisional Application No. 61/478,692, filed Apr. 25, 2011, andentitled “Stable Formulations for Parenteral Injection of PeptideDrugs”; of U.S. Provisional Application No. 61/553,388, filed Oct. 31,2011, and entitled “Formulations for the Treatment of Diabetes”; and ofU.S. Provisional Application No. 61/609,123, filed Mar. 9, 2012, andentitled “Formulations for the Treatment of Diabetes,” the entiredisclosures of which are herein incorporated by reference for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations and, moreparticularly, to pharmaceutical formulations of peptides having improvedstability and to methods of using such pharmaceutical formulations totreat various diseases, conditions and disorders.

BACKGROUND OF THE INVENTION

Diabetes is a serious health problem in modern society. Insulin is acritical treatment for both type I and type II diabetes. Studies overthe past two decades have demonstrated that tight metabolic control ofglucose through the use of insulin not only reduces the incidence, butalso delays the development of complications in people with type 1 andtype 2 diabetes. Unfortunately, the intensive insulin therapy requiredto achieve tight glucose control is also associated with a significantlyincreased risk of developing hypoglycemia or “low blood sugar.”

Symptoms of hypoglycemia vary greatly among patients, but typicallyinclude tremor, palpitations, irritability, anxiety, nervousness,hunger, tachycardia, headache and pallor. The symptoms typically subsideonce plasma glucose is restored to normal levels. If hypoglycemia is notreversed, a further decrease in plasma glucose can lead to depletion ofglucose in the central nervous system and associated neuroglycopenicsymptoms, such as difficulty in concentration, slurred speech, blurredvision, reduction in body temperature, behavioral changes and, if nottreated, unconsciousness, seizure and possibly death.

In general, hypoglycemia can be defined as minor to moderatehypoglycemia or as severe hypoglycemia as follows:

-   -   Minor to moderate hypoglycemia: Episodes that the patient can        self-treat, regardless of the severity of symptoms, or any        asymptomatic blood glucose measurements in which blood glucose        levels are less than 70 mg/dL (3.9 mmol/L).    -   Severe hypoglycemia: Operationally defined as an episode of        hypoglycemia that the patient cannot self-treat so that external        help is required. Typically, neuroglycopenic symptoms and        cognitive impairment begin at a blood glucose level of about 50        mg/dL (2.8 mmol/L).

Most episodes of minor to moderate hypoglycemia can be self-treatedrelatively easily by ingesting fast-acting carbohydrates such as glucosetablets or food (juice, soft drinks or sugary snacks). Severehypoglycemia, by definition, cannot be self-treated and thus requiresexternal intervention. If the patient can swallow and is cooperative, itis appropriate to use gels or products such as honey or jelly placedinside the cheek. If the patient is unable to swallow, glucagon, whichis injected subcutaneously or intramuscularly, is used to treat severehypoglycemia.

Glucagon is a naturally occurring peptide hormone that is 29 amino acidsin length and is secreted by the α cells of the pancreas. The principalfunction of glucagon is to maintain glucose production through bothglycogenolysis and gluconeogenesis, mostly mediated via the liver.Glucagon is the primary counter-regulatory hormone to insulin and isused as a first-line treatment of severe hypoglycemia in patients withdiabetes.

Numerous attempts have been made to create a glucagon rescue medicationfor treating severe hypoglycemia in emergency situations. Currently,there are two glucagon kits currently available in the United States,manufactured by Eli Lilly (Glucagon Emergency Kit) and Novo Nordisk(GlucaGen® HypoKit). Both products combine a vial of freeze-driedglucagon with a pre-filled syringe of aqueous diluent. The freeze-driedglucagon must be reconstituted using a complex procedure that isdifficult to use in an emergency situation. These products also providea large volume injection because glucagon is poorly soluble in water.Recently, attempts have been made to improve the stability of glucagonin an aqueous solution, to create more stable glucagon analogs and/or toimprove delivery of glucagon via powder injection.

Although some progress has been made, there still remains a need for amore user friendly glucagon rescue medication for treating severehypoglycemia in emergency situations. Such a glucagon rescue medicationwould need to be carried continuously by diabetics and/or theircaregivers and, thus, would need to be stable at nonrefrigeratedtemperatures (25-30° C.) for extended periods (>2 years). Ideally, itwould also need to be simple to administer for the general population,and not require excessive processing/reconstitution prior toadministration to the hypoglycemic patient. The glucagon rescuemedication would also need to be functional over a range oftemperatures, including temperatures ranging from 0° C.-30° C.

BRIEF SUMMARY OF THE INVENTION

To address such needs and others, the present invention provides astable glucagon rescue formulation as well as methods of using thisstable glucagon formulation to treat severe hypoglycemia.Advantageously, the glucagon is stabilized in the formulations of thepresent invention so as to allow for long-term storage and/or deliveryover a prolonged period of time. As such, the glucagon formulations ofthe present invention are stable at nonrefrigerated temperatures forextended periods of time, are simple to administer, without the need forreconstitution, and are functional over a range of temperatures,including temperatures ranging from 0° C.-30° C.

Importantly, the formulation technology of the present invention iswidely applicable for the delivery of numerous other peptides that, likeglucagon, have poor or limited stability and solubility in an aqueousenvironment. In fact, it is now clear that the formulation of peptideswith an aprotic polar solvent such as DMSO, NMP, ethyl acetate, or amixture thereof into high concentration, nonaqueous solutions is avaluable delivery platform for this important class of peptidetherapeutics. Additionally, the formulation technology of the presentinvention is widely applicable for the delivery of two or more peptidesin the same solution.

Thus, in one aspect, the present invention provides a stable formulationfor parenteral injection, the formulation comprising: (a) a peptide or asalt thereof, wherein the peptide has been dried in a non-volatilebuffer, and wherein the dried peptide has a pH memory that is aboutequal to the pH of the peptide in the non-volatile buffer; and (b) anaprotic polar solvent; wherein the moisture content of the formulationis less than 5%, and wherein the dried peptide maintains the pH memorythat is about equal to the pH of the peptide in the non-volatile bufferwhen the dried peptide is reconstituted in the aprotic polar solvent.

In another aspect, the present invention provides a stable formulationfor parenteral injection, the formulation comprising: (a) a firstpeptide or a salt thereof, wherein the first peptide has been dried in afirst non-volatile buffer, and wherein the first dried peptide has afirst pH memory that is about equal to the pH of the first peptide inthe first non-volatile buffer; (b) a second peptide or a salt thereof,wherein the second peptide has been dried in a second non-volatilebuffer, and wherein the second dried peptide has a second pH memory thatis about equal to the pH of the second peptide in the secondnon-volatile buffer; and (c) an aprotic polar solvent; wherein themoisture content of the formulation is less than 5%, wherein the firstdried peptide maintains the first pH memory that is about equal to thepH of the first peptide in the first non-volatile buffer when the firstdried peptide is reconstituted in the aprotic polar solvent, and whereinthe second dried peptide maintains the second pH memory that is aboutequal to the pH of the second peptide in the second non-volatile bufferwhen the second dried peptide is reconstituted in the aprotic polarsolvent.

In another aspect, the present invention provides a stable formulationfor parenteral injection, the formulation comprising: a peptide or asalt thereof (such as a hydrochloride or acetate salt thereof); and anaprotic polar solvent, wherein the moisture content of the formulationis less than 5%.

The stable formulations described herein are useful for the parenteralinjection of any peptide that has limited or poor stability orsolubility in an aqueous environment. Thus, in some embodiments, thepeptide (or each of the first and second peptides) or salt thereof isselected from the group consisting of glucagon, pramlintide, insulin,leuprolide, an LHRH agonist, parathyroid hormone (PTH), amylin,botulinum toxin, hematide, an amyloid peptide, cholecystikinin, aconotoxin, a gastric inhibitory peptide, an insulin-like growth factor,a growth hormone releasing factor, an anti-microbial factor, glatiramer,glucagon-like peptide-1 (GLP-1), a GLP-1 agonist, exenatide, analogsthereof, and mixtures thereof. In a preferred embodiment, the peptide isglucagon or a glucagon analog or a glucagon peptidomimetic. In anotherembodiment, the peptide is parathyroid hormone. In yet anotherembodiment, the peptide is leuprolide. In still another embodiment, thepeptide is glatiramer. In yet another embodiment, the first peptide ispramlintide and the second peptide is insulin. In still anotherembodiment, the first peptide is glucagon and the second peptide isexenatide.

The peptide (or, in embodiments where the formulation comprises two ormore peptides, each of the peptides) is mixed with a non-volatile bufferand dried to a dry peptide powder. Suitable non-volatile buffersinclude, but are not limited to, glycine buffers, citrate buffers,phosphate buffers, and mixtures thereof. In one preferred embodiment,the non-volatile buffer is a glycine buffer. In another preferredembodiment, the non-volatile buffer is a mixture of citrate buffer andphosphate buffer. In some embodiments, wherein the formulation comprisestwo or more peptides, the first non-volatile buffer and the secondnon-volatile buffer are the same. In some embodiments, wherein theformulation comprises two or more peptides, the first non-volatilebuffer and the second non-volatile buffer are different.

In some formulations of the present invention, the peptide is mixed witha non-volatile buffer and a stabilizing excipient, and then dried to adry peptide powder. Suitable stabilizing excipients include, but are notlimited to, sugars, starches, and mixtures thereof. In some embodiments,the sugar is trehalose. In some embodiments, the starch is hydroxyethylstarch (HES). In some embodiments, the stabilizing excipient is presentin the formulation in an amount ranging from about 1% (w/v) to about 60%(w/v), from about 1% (w/v) to about 50% (w/v), from about 1% (w/v) toabout 40% (w/v), from about 1% (w/v) to about 30% (w/v), from about 1%(w/v) to about 20% (w/v), from about 5% (w/v) to about 60% (w/v), fromabout 5% (w/v) to about 50% (w/v), from about 5% (w/v) to about 40%(w/v), from about 5% (w/v) to about 30% (w/v), from about 5% (w/v) toabout 20% (w/v), from about 10% (w/v) to about 60% (w/v), from about 10%(w/v) to about 50% (w/v), from about 10% (w/v) to about 40% (w/v), fromabout 10% (w/v) to about 30% (w/v), or from about 10% (w/v) to about 20%(w/v). In some embodiments, wherein the formulation comprises twopeptides, both of the first peptide in the first non-volatile buffer andthe second peptide in the second non-volatile buffer further comprise astabilizing excipient, and the stabilizing excipient with the firstpeptide in the first non-volatile buffer and the stabilizing excipientwith the second peptide in the second non-volatile buffer are the same.In other embodiments, wherein the formulation comprises two peptides,both of the first peptide in the first non-volatile buffer and thesecond peptide in the second non-volatile buffer further comprise astabilizing excipient, and the stabilizing excipient with the firstpeptide in the first non-volatile buffer and the stabilizing excipientwith the second peptide in the second non-volatile buffer are different.

Once the peptide or peptides and the non-volatile buffer or thepeptide(s), the non-volatile buffer and the stabilizing excipient aredried to a powder, the dried peptide powder is dissolved orreconstituted in an aprotic polar solvent. Examples of aprotic polarsolvents include, but are not limited to, the following:dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate,n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), propylenecarbonate, and mixtures thereof. Dimethylsulfoxide (DMSO), n-methylpyrrolidone (NMP), ethyl acetate, and mixtures of one or more of DMSO,NMP, and ethyl acetate are particularly preferred aprotic polarsolvents. In a preferred embodiment, the aprotic polar solvent is DMSO.In another preferred embodiment, the aprotic polar solvent is a mixtureof DMSO and NMP. In yet another preferred embodiment, the aprotic polarsolvent is a mixture of DMSO and ethyl acetate.

In some embodiments, the peptide or peptides are reconstituted in amixture of an aprotic polar solvent (e.g., dimethylsulfoxide (DMSO),dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP),dimethylacetamide (DMA), propylene carbonate, or mixtures thereof) and aco-solvent that depresses the freezing point of the formulation. In someembodiments, the co-solvent depresses the freezing point of theformulation by at least about 5° C., at least about 10° C., at leastabout 15° C., or at least about 20° C. In some embodiments, theco-solvent depresses the freezing point of the formulation to about 3°C., to about 2° C., to about 1° C., or to about 0° C. or below. In someembodiments, the co-solvent is a polar protic solvent. In preferredembodiments, the co-solvent is selected from ethanol, propylene glycol(PG), glycerol, and mixtures thereof. In some embodiments, theco-solvent is present in the formulation in an amount ranging from about10% (w/v) to about 50% (w/v), from about 10% (w/v) to about 40% (w/v),from about 10% (w/v) to about 30% (w/v), from about 10% (w/v) to about25% (w/v), from about 15% (w/v) to about 50% (w/v), from about 15% (w/v)to about 40% (w/v), from about 15% (w/v) to about 30% (w/v), or fromabout 15% (w/v) to about 25% (w/v).

Importantly, the formulations of the present invention have very littleresidual moisture and, thus, the peptides in such formulations remainstable over extended periods of time. In preferred embodiments, themoisture content of the formulation of the present invention is lessthan about 4%, preferably, less than about 3%, preferably, less thanabout 2%, and even more preferably, less than about 1%, preferably, lessthan about 0.5%, preferably, less than about 0.25%, preferably, lessthan about 0.2%, preferably, less than about 0.15%, or preferably, lessthan about 0.1%. In other preferred embodiments, the moisture content ofthe formulation of the present invention is from about 0.01% to about4%, preferably, from about 0.01% to about 3%, preferably, from about0.01% to about 2%, preferably, from about 0.01% to about 1%, preferably,from about 0.1% to about 4%, preferably, from about 0.1% to about 3%,preferably, from about 0.1% to about 2%, preferably, from about 0.1% toabout 1%, preferably, from about 0.25% to about 4%, preferably, fromabout 0.25% to about 3%, preferably, from about 0.25% to about 2%,preferably, from about 0.25% to about 1%, or preferably, from about 0.5%to about 1%.

When the peptide is mixed with a nonvolatile buffer, the nonvolatilebuffer is selected such that the peptide has a pH of maximal stability,maximal solubility, and minimal degradation in the aqueous environment.Once dried, the peptide will have a pH memory of maximal stability,maximal solubility, and minimal degradation and will retain that pHmemory when dissolved in or reconstituted in the aprotic polar solvent.As such, in preferred embodiments, the peptide in the formulation willhave a pH memory of about 2.0 to about 3.0 to ensure maximal stability,maximal solubility, and minimal degradation. In other embodiments, thepeptide in the formulation will have a pH memory of about 3.0 to about5.0 to ensure maximal stability, maximal solubility, and minimaldegradation. In other embodiments, the peptide in the formulation willhave a pH memory of about 4.0 to about 5.0 to ensure maximal stability,maximal solubility, and minimal degradation. In yet other embodiments,the peptide will have a pH memory of about 4.0 to about 6.0 to ensuremaximal stability, maximal solubility, and minimal degradation. In yetother embodiments, the peptide will have a pH memory of about 6.0 toabout 8.0 to ensure maximal stability, maximal solubility, and minimaldegradation. In some embodiments, wherein the formulation comprises twopeptides, the first peptide has a pH memory of about 4.0 to about 6.0 toensure maximal stability, maximal solubility, and minimal degradation,and the second peptide has a pH memory of about 1.5 to about 2.5, or ofabout 6.0 to about 8.0, to ensure maximal stability, maximal solubility,and minimal degradation. In some embodiments, wherein the formulationcomprises two peptides, the first peptide has a pH memory of about 3.0to about 5.0 to ensure maximal stability, maximal solubility, andminimal degradation, and the second peptide has a pH memory of about 1.5to about 2.5, or of about 6.0 to about 8.0, to ensure maximal stability,maximal solubility, and minimal degradation. In other embodiments,wherein the formulation comprises two peptides, the first peptide has apH memory of about 2.0 to about 3.0 to ensure maximal stability, maximalsolubility, and minimal degradation, and the second peptide has a pHmemory of about 4.0 to about 5.0 to ensure maximal stability, maximalsolubility, and minimal degradation. It will be readily apparent to oneof skill in the art how to determine the optimal pH for obtaining apeptide having maximal stability, maximal solubility, and minimaldegradation.

Any suitable dosage of peptide or peptides can be formulated in thestable formulations of the present invention. Generally, the peptide(or, in embodiments comprising two or more peptides, each of thepeptides) is present in the formulation in an amount ranging from about0.5 mg/mL to about 100 mg/mL. In some embodiments, the peptide ispresent in the formulation in an amount ranging from about 10 mg/mL toabout 60 mg/mL. In other embodiments, the peptide is present in theformulation in an amount ranging from about 20 mg/mL to about 50 mg/mL.In still other embodiments, the peptide is present in the formulation inan amount ranging from about 5 mg/mL to about 15 mg/mL. In yet otherembodiments, the peptide is present in the formulation in an amountranging from about 0.5 mg/mL to about 2 mg/mL. In yet other embodiments,the peptide is present in the formulation in an amount ranging fromabout 1 mg/mL to about 50 mg/mL. Again, it will be readily apparent tothose of skill that the peptide dosage can be varied depending on thepeptide used and the disease, disorder or condition to be treated.

In some embodiments, the formulations of the present invention furthercomprise an antioxidant. In other embodiments, the formulations furthercomprise a chelator. In still other embodiments, the formulations of thepresent invention further comprise a preservative.

In another aspect, the present invention provides a method for treatinga disease, condition or disorder that may be treated, alleviated, orprevented by administering to a subject a stable peptide formulation asdescribed herein in an amount effective to treat, alleviate or preventthe disease, condition, or disorder. In some embodiments, the disease,condition, or disorder is hypoglycemia. In some embodiments, wherein thedisease, condition, or disorder is hypoglycemia, the method comprisesadministering a stable glucagon formulation of the present invention inan amount effective to treat the hypoglycemia. In some embodiments, thedisease, condition, or disorder is diabetes. In some embodiments,wherein the disease, condition, or disorder is diabetes, the methodcomprises administering a stable insulin and pramlintide formulation ofthe present invention in an amount effective to treat the diabetes.

In yet another aspect, the present invention provides a process formaking a stable formulation for parenteral injection, the processcomprising: drying a peptide and a nonvolatile buffer to a dry peptidepowder; and reconstituting the dry peptide powder with an aprotic polarsolvent, thereby making the stable formulation, wherein the moisturecontent of the stable formulation is less than 5%. In some embodiments,the dried peptide powder has a pH memory that is about equal to the pHof the peptide in the non-volatile buffer, and the dried peptide powdermaintains the pH memory that is about equal to the pH of the peptide inthe non-volatile buffer when the dried peptide powder is reconstitutedin the aprotic polar solvent.

In still another aspect, the present invention provides kits fortreating a disease, condition or disorder, the kit comprising: a stableformulation comprising one or more peptides or salts thereof, whereinthe peptide(s) has been dried in a non-volatile buffer, and wherein thedried peptide(s) has a pH memory that is about equal to the pH of thepeptide(s) in the non-volatile buffer; and an aprotic polar solvent;wherein the moisture content of the formulation is less than 5%, andwherein the dried peptide(s) maintains the pH memory that is about equalto the pH of the peptide(s) in the non-volatile buffer when the driedpeptide(s) is reconstituted in the aprotic polar solvent; and a syringefor administration of the stable formulation to the subject.

In some embodiments, the kit is for treating hypoglycemia and the stableformulation comprises a glucagon formulation as described herein. Insome embodiments, the kit is for treating diabetes and the stableformulation comprises an insulin and pramlintide formulation asdescribed herein. In some embodiments, the syringe is part of a peninjection device, an auto-injector device or a pump. In some embodiment,the syringe is prefilled with the stable formulation. In someembodiments, the kit further comprises instructions, wherein theinstructions direct the administration of the stable formulation totreat the subject in need thereof.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates plasma glucagon levels after injection offreeze-dried glucagon-glycine-trehalose dissolved in DMSO or NMP.

FIG. 2 illustrates blood glucose levels after injection of freeze-driedglucagon-glycine-trehalose dissolved in DMSO or NMP.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Peptides can degrade via a number of different mechanisms, includingdeamidation, oxidation, hydrolysis, disulfide interchange andracemization. Further, water acts as a plasticizer, which facilitatesunfolding of protein molecules and irreversible molecular aggregation.Therefore, in order to provide a peptide formulation that is stable overtime at ambient or physiological temperatures, a nonaqueous orsubstantially nonaqueous peptide formulation is generally required.

Reduction of aqueous peptide formulations to dry powdered formulationsis one way to increase the stability of pharmaceutical peptideformulations. For example, peptide formulations can be dried usingvarious techniques, including spray-drying, lyophilization orfreeze-drying, and desiccation. The dry powder peptide formulationsachieved by such techniques exhibit significantly increased stabilityover time at ambient or even physiological temperatures.

The present invention is based, in part, on the surprising discoverythat a stable peptide formulation (e.g., a stable glucagon rescueformulation) can be readily prepared by first freeze-drying one or morepeptides (e.g., a glucagon peptide) in a non-volatile buffer to a drypeptide powder. The dried peptide has a defined “pH memory” of the pH ofthe peptide in the non-volatile buffer from which the peptide was dried.Once dried, the resulting peptide powder, e.g., the freeze-driedglucagon, is dissolved in an aprotic polar solvent, thereby forming astable formulation, wherein the moisture content of the formulation isless than 5% and, preferably, less than 4%, less than 3%, less than 2%,less than 1%, less than 0.5%, less than 0.25%, less than 0.15%, or lessthan 0.1%. The dried peptide maintains its defined pH memory whenreconstituted in the aprotic polar solvent, i.e., the pH of the peptidewhen reconstituted in the aprotic polar solvent is about equal to the pHof the peptide in the non-volatile buffer from which it was dried.Advantageously, once prepared, the formulation (e.g., the glucagonformulation) is stable for extended periods of time, is ready for usewithout the need for reconstitution, and is functional over a range oftemperatures.

Importantly, the formulation technology of the present invention iswidely applicable for the delivery of numerous other peptides that, likeglucagon, have poor or limited stability and solubility in an aqueousenvironment. In fact, it is now clear that formulation of peptides withan aprotic polar solvent (e.g., DMSO, NMP, ethyl acetate, or a mixturethereof) into high concentration, nonaqueous solutions is an invaluabledelivery platform for an important class of therapeuticagents—therapeutic peptides. The stable formulations described hereinadvantageously promote uniform delivery of the peptide drugs and provideadditional shelf stability against aggregation, oxidation, andhydrolysis related degradation pathways.

In certain preferred embodiments, the stable formulations describedherein preserve the peptide drugs in a stable form for a prolongedperiod of time, e.g., for a period of time sufficient to provide adesired shelf life of the formulation without unacceptable levels ofdegradation of the therapeutic agent prior to use. A desired property ofthe injectable formulations is that they be nonaqueous and nonreactivewith respect to the peptide. In such embodiments, it is possible tostore the injectable formulations directly in the injection deviceitself.

The stable injectable formulations of the present invention contain thenecessary delivered dose of therapeutic peptide or peptides (e.g., thedose required for drug therapy) and are preferably low volume. Forexample, in some embodiments an injectable formulation comprising atherapeutic dose of a peptide (e.g., glucagon) has a volume of at leastabout 1.0 microliters (the lower limit being a function of the fillingequipment), more preferably from about 10 milliliters to about 250microliters. The delivery of a therapeutic dose of peptide at a lowvolume is accomplished in certain preferred embodiments by concentratingthe dose of the therapeutic peptide or peptides (e.g., glucagon) in astable form in a suitable aprotic polar solvent for injection inaccordance with the invention.

Furthermore, the stable formulations of the present invention aresuitable for administration without requiring dilution prior toinjection. Many currently available therapeutic peptide and vaccineproducts are produced in a solid particulate form to promote stabilitywhile on the shelf. These formulations are diluted prior to injection insterile water, phosphate buffer solution, or isotonic saline. Incontrast, in certain preferred embodiments of the present invention, thetherapeutic peptide is concentrated using the particle preparationprocessing techniques (e.g., spray drying, lyophilization, etc.)routinely employed by the pharmaceutical industry to prepareformulations for injection. In preferred embodiments, therapeuticdosages of peptide drugs are achieved by dissolving the peptides, whichhave first been freeze-dried with a non-volatile buffer (and optionallyadditional components such as a stabilizing excipient) to a dried powderhaving very little residual moisture content. Once prepared, the driedpeptide powder is dissolved in an aprotic polar solvent, such as DMSO,NMP, ethyl acetate, or blends of these solvents. Thus, in accordancewith the goals of the present invention, the low volume, stableformulations of the present invention are injected, infused, orotherwise administered into an animal (e.g., human patient), withoutfirst diluting the formulation prior to injection as required by mostreconstitution products. As such, in preferred embodiments, the lowvolume formulations of the present invention are administrable withoutbeing first being diluted, or reconstituted, or refrigerated.

II. Definitions

For purposes of the present disclosure, the following terms have thefollowing meanings:

The term “therapeutic agent” encompasses peptide compounds together withpharmaceutically acceptable salts thereof. Useful salts are known tothose skilled in the art and include salts with inorganic acids, organicacids, inorganic bases, or organic bases. Therapeutic agents useful inthe present invention are those peptide compounds that affects adesired, beneficial, and often pharmacological, effect uponadministration to a human or an animal, whether alone or in combinationwith other pharmaceutical excipients or inert ingredients.

The terms “peptide,” “polypeptide” and/or “peptide compound” referpolymers of up to about 80 amino acid residues bound together by amide(CONH) linkages. Analogs, derivatives, agonists, antagonists andpharmaceutically acceptable salts of any of the peptide compoundsdisclosed here are included in these terms. The terms also includepeptides and/or peptide compounds that have D-amino acids, modified,derivatized or normaturally occurring amino acids in the D- orL-configuration and/or peptomimetic units as part of their structure.

The term “pharmaceutically acceptable carrier” means a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering a peptidecompound of the present invention to a mammal such as an animal orhuman. In a presently preferred embodiment, the pharmaceuticallyacceptable carrier is an aprotic polar solvent.

The term “aprotic polar solvent” means a polar solvent that does notcontain acidic hydrogen and does not act as a hydrogen bond donor.Examples of aprotic polar solvents include, but are not limited to,dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate,n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), and propylenecarbonate. The term aprotic polar solvent also encompasses mixtures oftwo or more aprotic polar solvents, e.g., a mixture of two or more ofdimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate,n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), and propylenecarbonate.

The term “pharmaceutically acceptable” ingredient, excipient orcomponent is one that is suitable for use with humans and/or animalswithout undue adverse side effects (such as toxicity, irritation andallergic response) commensurate with a reasonable benefit/risk ratio.

The term “chemical stability” means that with respect to the therapeuticagent, an acceptable percentage of degradation products produced bychemical pathways such as oxidation or hydrolysis is formed. Inparticular, a formulation is considered chemically stable if no morethan about 20% breakdown products are formed after one year of storageat the intended storage temperature of the product (e.g., roomtemperature); or storage of the product at 30° C./60% relative humidityfor one year; or storage of the product at 40° C./75% relative humidityfor one month, and preferably three months. In some embodiments, achemically stable formulation has less than 20%, less than 15%, lessthan 10%, less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% breakdown products formed after an extended period ofstorage at the intended storage temperature of the product.

The term “physical stability” means that with respect to the therapeuticagent, an acceptable percentage of aggregates (e.g., dimers, trimers andlarger forms) is formed. In particular, a formulation is consideredphysically stable if no more that about 15% aggregates are formed afterone year of storage at the intended storage temperature of the product(e.g., room temperature); or storage of the product at 30° C./60%relative humidity for one year; or storage of the product at 40° C./75%relative humidity for one month, and preferably three months. In someembodiments, a physically stable formulation has less than less than15%, less than 10%, less than 5%, less than 4%, less than 3%, less than2%, or less than 1% aggregates formed after an extended period ofstorage at the intended storage temperature of the product.

The term “stable formulation” means that at least about 65% chemicallyand physically stable therapeutic agent remains after two months ofstorage at room temperature. Particularly preferred formulations arethose in which at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% chemically and physically stable therapeutic agentremains under these storage conditions. Especially preferred stableformulations are those which do not exhibit degradation aftersterilizing irradiation (e.g., gamma, beta or electron beam).

The phrase “consisting essentially of” is used herein to exclude anyelements that would substantially alter the essential properties of thestable formulations to which the phrase refers.

The term “bioavailability” is defined for purposes of the presentinvention as the extent to which the therapeutic agent, such as apeptide compound, is absorbed from the formulation.

The term “systemic” means, with respect to delivery or administration ofa therapeutic agent, such as a peptide compound, to a subject, thattherapeutic agent is detectable at a biologically significant level inthe blood plasma of the subject.

The term “controlled release” is defined for purposes of the presentinvention as the release of the therapeutic agent at such a rate thatblood (e.g., plasma) concentrations are maintained within thetherapeutic range, but below toxic concentrations over a period of timeof about one hour or longer, preferably 12 hours or longer.

The term “parenteral injection” refers to the administration oftherapeutic agents, such as peptide compounds, via injection under orthrough one or more layers of skin or mucus membranes of an animal, suchas a human. Standard parenteral injections are given into theintradermal, subcutaneous, or intramuscular region of an animal, e.g., ahuman patient. In some embodiments, a deep location is targeted forinjection of a therapeutic agent as described herein.

The terms “treat” or “treatment” refer to delaying the onset of,retarding or reversing the progress of, or alleviating or preventingeither the disease or condition to which the term applies, or one ormore symptoms of such disease or condition.

The terms “patient,” “subject,” or “individual” interchangeably refer toa mammal, for example, a human or a non-human mammal, e.g., a primate,dog, cat, bovine, ovine, porcine, equine, mouse, rat, hamster, rabbit,or guinea pig.

III. Stable Peptide Formulations

In one aspect, the present invention provides a stable formulation forparenteral injection. Advantageously, once prepared, the formulation isstable for extended periods of time, is ready for use without the needfor reconstitution, and is functional over a range of temperatures.Furthermore, the stable formulation of the present invention is usefulfor the parenteral injection of any peptide that has limited or poorstability or solubility in an aqueous environment. In some embodiments,the formulations of the present invention increase the physicalstability of the peptide or peptides of the formulation, for example, bypreventing or decreasing the formation of aggregates of the peptide orpeptides.

In some embodiments, the formulation comprises: (a) a peptide or a saltthereof, wherein the peptide has been dried in a non-volatile buffer,and wherein the dried peptide has a pH memory that is about equal to thepH of the peptide in the non-volatile buffer; and (b) an aprotic polarsolvent; wherein the moisture content of the formulation is less than5%, and wherein the dried peptide maintains the pH memory that is aboutequal to the pH of the peptide in the non-volatile buffer when the driedpeptide is reconstituted in the aprotic polar solvent.

In some embodiments, the formulation comprises: (a) a first peptide or asalt thereof, wherein the first peptide has been dried in a firstnon-volatile buffer, and wherein the first dried peptide has a first pHmemory that is about equal to the pH of the first peptide in the firstnon-volatile buffer; (b) a second peptide or a salt thereof, wherein thesecond peptide has been dried in a second non-volatile buffer, andwherein the second dried peptide has a second pH memory that is aboutequal to the pH of the second peptide in the second non-volatile buffer;and (c) an aprotic polar solvent; wherein the moisture content of theformulation is less than 5%, wherein the first dried peptide maintainsthe first pH memory that is about equal to the pH of the first peptidein the first non-volatile buffer when the first dried peptide isreconstituted in the aprotic polar solvent, and wherein the second driedpeptide maintains the second pH memory that is about equal to the pH ofthe second peptide in the second non-volatile buffer when the seconddried peptide is reconstituted in the aprotic polar solvent.

In some embodiments, the formulation consists essentially of: (a) apeptide or a salt thereof, wherein the peptide has been dried in anon-volatile buffer, and wherein the dried peptide has a pH memory thatis about equal to the pH of the peptide in the non-volatile buffer; and(b) an aprotic polar solvent; wherein the moisture content of theformulation is less than 5%, and wherein the dried peptide maintains thepH memory that is about equal to the pH of the peptide in thenon-volatile buffer when the dried peptide is reconstituted in theaprotic polar solvent.

A. Peptides

The stable formulations of the present invention comprise one, two,three, four, or more peptides or salts, analogs, and/or mixturesthereof. Peptides (as well as salts thereof) suitable for use in theformulations of the present invention include, but are not limited to,glucagon, pramlintide, insulin, leuprolide, anluteinizing-hormone-releasing hormone (LHRH) agonist, parathyroidhormone (PTH), amylin, botulinum toxin, hematide, an amyloid peptide,cholecystikinin, gastric inhibitory peptide, an insulin-like growthfactor, growth hormone releasing factor, anti-microbial factor,glatiramer, glucagon-like peptide-1 (GLP-1), a GLP-1 agonist, exenatide,analogs thereof, and mixtures thereof. In some embodiments, the peptideis a hydrochloride salt or an acetate salt.

In a preferred embodiment, the peptide is glucagon or a glucagon analogor peptidomimetic, or a salt thereof (e.g., glucagon acetate). Inanother embodiment, the peptide is parathyroid hormone. In yet anotherembodiment, the peptide is leuprolide. In still another embodiment, thepeptide is glatiramer. In other embodiments, the peptide is amylin or anamylinomimetic (e.g., pramlintide). In still other embodiments, thepeptide is insulin or an insulin analog (e.g., Lispro). In someembodiments, the insulin or insulin analog preparation is a low-zinc orzinc-free preparation.

In some embodiments, the formulation comprises two peptides, wherein thefirst peptide is amylin or an amylinomimetic and the second peptide isinsulin or an insulin analog. In some embodiments, the first peptide ispramlintide and the second peptide is insulin. In some embodiments, thefirst peptide is pramlintide and the second peptide is a low-zincinsulin preparation or a zinc-free insulin preparation.

In some embodiments, the formulation comprises two peptides, wherein thefirst peptide is glucagon and the second peptide is a glucagon-likepeptide-1 (GLP-1) or a GLP-1 analog or agonist (e.g., exenatide). Insome embodiments, the first peptide is glucagon and the second peptideis GLP-1. In some embodiments, the first peptide is glucagon and thesecond peptide is exenatide.

Any suitable dosage of peptide or peptides can be administered using theformulations of the present invention. The dosage administered will, ofcourse, vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular peptide, salt, or combination thereof;the age, health, or weight of the subject; the nature and extent ofsymptoms; the metabolic characteristics of the therapeutic agent andpatient, the kind of concurrent treatment; the frequency of treatment;or the effect desired. Generally, the peptide (or, wherein the stableformulation comprises two or more peptides, each of the peptides) ispresent in the formulation in an amount ranging from about 0.5 mg/mL toabout 100 mg/mL (e.g., about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100mg/mL).

In some embodiments, the peptide is present in the formulation in anamount ranging from about 0.5 mg/mL to about 60 mg/mL. In someembodiments, the peptide is present in the formulation in an amountranging from about 10 mg/mL to about 50 mg/mL. In other embodiments, thepeptide is present in the formulation in an amount ranging from about 20mg/mL to about 50 mg/mL. In still other embodiments, the peptide ispresent in said formulation in an amount ranging from about 5 mg/mL toabout 15 mg/mL. In yet other embodiments, the peptide is present in theformulation in an amount ranging from about 0.5 mg/mL to about 2 mg/mL.Again, it will be readily apparent to those of skill that the peptidedosage can be varied depending on the peptide used and the disease,disorder or condition to be treated.

In preferred embodiments, the peptide is mixed with a non-volatilebuffer, and optionally a stabilizing excipient, and then dried to a drypeptide powder. In embodiments where the stable formulation comprisestwo or more peptides, each of the peptides is separately mixed with anon-volatile buffer, and optionally a stabilizing excipient, and thendried to a dry peptide powder. Peptides are susceptible to hydrolysis atbonds with asparagine residues and oxidation of methionine, so the useof non-volatile buffers in the formulations of the present inventionbeneficially affects chemical stability. As described in further detailbelow, while pH is not relevant in an aprotic polar solvent, the chargeprofile of a peptide in an aprotic polar solvent will affect itsstability. The charge profile of a peptide in an aprotic polar solventwill be a function of the pH of the aqueous solution from which it waspreviously dried, i.e., there is a “pH memory” after dissolution orreconstitution in an aprotic polar solvent. To achieve the desiredcharge profile for a peptide dissolved in an aprotic polar solvent, thepeptide is dried from a buffered aqueous solution at the pH that yieldsthe optimal stability, optimal solubility, and minimal degradation inthe aprotic polar solvent.

As such, non-volatile buffers that are useful in the formulationsdescribed herein are those that are helpful in establishing a pH ofmaximum stability, maximum solubility, and minimal degradation as wellas those that are helpful in removing residual moisture or water contentfrom the dried peptide powder. Non-volatile buffers include thosebuffers that will not evaporate away in a manner similar to water upondrying/lyophilization. Suitable non-volatile buffers include, forexample, glycine buffers, citrate buffers, phosphate buffers, andmixtures thereof. In some embodiments, the non-volatile buffer is aglycine buffer or a citrate buffer. In some embodiments, thenon-volatile buffer is a glycine buffer. In some embodiments, thenon-volatile buffer is a mixture of glycine buffer and citrate buffer.In some embodiments, the non-volatile buffer is a mixture of citratebuffer and phosphate buffer.

B. Stabilizing Excipients

In certain preferred embodiments, the formulations described herein maybe further stabilized to ensure the stability of the peptideincorporated therein. In some embodiments, the stability of theinjectable formulation is enhanced by the inclusion of one or morestabilizing agents or stabilizing excipients into the formulation priorto the drying of the peptide or peptides. In other embodiments, thestability of the injectable formulation is enhanced by reconstitutingthe dried peptide or peptides with a stabilizing agent or stabilizingexcipient in the aprotic polar solvent.

In some embodiments, the stabilizing excipient is a cryoprotectant. Asshown below in the Examples section, the addition of a cryoprotectant,such as trehalose, protects the peptide formulations of the presentinvention against instability associated with freeze-thaw cycles.Furthermore, it has been shown herein that the addition of thecryoprotectant trehalose also promotes enhanced thawing of a frozenpeptide formulation. This property of enhanced thawing is surprisinglyadvantageous, particularly in emergency medical situations, such as asevere hypoglycemia episode, wherein a peptide formulation of thepresent invention is frozen and needs to be administered quickly. Thus,in another aspect of the present invention, the stable formulation hasan improved freeze-thaw stability, an enhanced thawing rate, and/or anenhanced thawing profile.

In some embodiments, the stabilizing excipient is selected from sugars,starches, sugar alcohols, and mixtures thereof. Examples of suitablesugars for stabilizing excipients include, but are not limited to,trehalose, glucose, sucrose, etc. Examples of suitable starches forstabilizing excipients include, but are not limited to, hydroxyethylstarch (HES). Examples of suitable sugar alcohols for stabilizingexcipients include, but are not limited to, mannitol and sorbitol. Insome embodiments, the at least one stabilizing excipient (e.g., a sugar,a starch, a sugar alcohol, or a mixture thereof) is capable of enhancingthe stability of the peptide during a freeze-thawing process, enhancingthe thawing rate of the formulation, or enhancing the thawing profile ofthe formulation.

In some embodiments, the stabilizing excipient is present in theformulation in an amount ranging from about 1% (w/v) to about 60% (w/v),from about 1% (w/v) to about 50% (w/v), from about 1% (w/v) to about 40%(w/v), from about 1% (w/v) to about 30% (w/v), from about 1% (w/v) toabout 20% (w/v), from about 5% (w/v) to about 60% (w/v), from about 5%(w/v) to about 50% (w/v), from about 5% (w/v) to about 40% (w/v), fromabout 5% (w/v) to about 30% (w/v), from about 5% (w/v) to about 20%(w/v), from about 10% (w/v) to about 60% (w/v), from about 10% (w/v) toabout 50% (w/v), from about 10% (w/v) to about 40% (w/v), from about 10%(w/v) to about 30% (w/v), or from about 10% (w/v) to about 20% (w/v). Insome embodiments, the stabilizing excipient is present in theformulation in an amount that is about 1%, about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, or about 60% (w/v).

In formulations comprising two or more peptides, in some embodimentseach of the peptides are dried in a mixture comprising a non-volatilebuffer and a stabilizing excipient. The mixtures of the non-volatilebuffer and the stabilizing excipient may be the same for each peptide,or the non-volatile buffer, the stabilizing excipient, or both thenon-volatile buffer and stabilizing excipient that is used for dryingeach peptide may be different. In other embodiments, some but not all ofthe peptides may be dried in a mixture comprising a non-volatile bufferand a stabilizing excipient, while other peptides may be dried in anon-volatile buffer in the absence of a stabilizing excipient.

In some embodiments, the formulation further comprises additionalstabilizing agents including, for example, antioxidants, chelators andpreservatives. Examples of suitable antioxidants include, but are notlimited to, ascorbic acid, cysteine, methionine, monothioglycerol,sodium thiosulphate, sulfites, BHT, BHA, ascorbyl palmitate, propylgallate, N-acetyl-L-cysteine (NAC), and Vitamin E. Examples of suitablechelators include, but are not limited to, EDTA, tartaric acid and saltsthereof, glycerin, and citric acid and salts thereof. Examples ofsuitable preservatives include, but are not limited to, benzyl alcohols,methyl parabens, propyl parabens, and mixtures thereof.

In some embodiments, the formulation further comprises a stabilizingpolyol. Such formulations and materials are described, for example, inU.S. Pat. Nos. 6,290,991 and 6,331,310, the contents of each of whichare incorporated by reference herein.

C. Reconstitution of Dried Peptides

In the stable formulations of the present invention, once the peptideand non-volatile buffer (and optionally the stabilizing excipient) aredried to a powder, or where the formulation comprises two or morepeptides, once each of the peptide and non-volatile buffer (eachoptionally also comprising a stabilizing excipient) is dried to apowder, the dried peptide powder is, or the dried peptide powders are,dissolved or reconstituted in an aprotic polar solvent. In someembodiments, the aprotic polar solvent is selected fromdimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate,n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), propylenecarbonate, and mixtures thereof. In some embodiments, the aprotic polarsolvent is a mixture of two or more of dimethylsulfoxide (DMSO),dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP),dimethylacetamide (DMA), and propylene carbonate. Dimethylsulfoxide(DMSO), ethyl acetate, and n-methyl pyrrolidone (NMP) are particularlypreferred aprotic polar solvents, each of which is a biocompatiblesolvent. In some embodiments, the aprotic polar solvent isdimethylsulfoxide (DMSO). In other embodiments, the aprotic polarsolvent is n-methyl pyrrolidone (NMP). In other embodiments, the aproticpolar solvent is a mixture of dimethylsulfoxide (DMSO) and n-methylpyrrolidone (NMP). In still other embodiments, the aprotic polar solventis a mixture of dimethylsulfoxide (DMSO) and ethyl acetate. In someembodiments, the dried peptide powder is reconstituted in an aproticpolar solvent that is “neat,” i.e., that does not contain a co-solvent.In some embodiments, the dried peptide powder is reconstituted in asolution that comprises an aprotic polar solvent and that does notcontain water as a co-solvent.

In some embodiments, the formulations of the present invention furthercomprise at least one co-solvent that depresses the freezing point ofthe formulation. The co-solvent is a polar protic solvent. In someembodiment, the co-solvent is selected from ethanol, propylene glycol(PG), glycerol, and mixtures thereof. In some embodiments, theco-solvent is ethanol or propylene glycol (PG). The co-solvent may bepresent in the formulation in an amount ranging from about 10% (w/v) toabout 50% (w/v), e.g., about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, or about 50% (w/v). In someembodiments, the co-solvent is present in the formulation in an amountranging from about 10% (w/v) to about 50% (w/v), from about 10% (w/v) toabout 40% (w/v), from about 10% (w/v) to about 30% (w/v), from about 10%(w/v) to about 25% (w/v), from about 15% (w/v) to about 50% (w/v), fromabout 15% (w/v) to about 40% (w/v), from about 15% (w/v) to about 30%(w/v), or from about 15% (w/v) to about 25% (w/v). In some embodiments,the at least one co-solvent depresses the freezing point of theformulation by at least 5° C., at least 10° C., at least 15° C., atleast 20° C. or more as compared to an otherwise identical formulationthat does not comprise the co-solvent. In some embodiments, the at leastone co-solvent depresses the freezing point of the formulation to about3° C., to about 2° C., to about 1° C., or to about 0° C. or below.

D. Moisture Content

The formulations of the present invention have very little residualmoisture and, thus, the peptides in such formulations remain stable overextended periods of time. In some embodiments, the stable formulationsof the present invention have a moisture content that is less than 5%.In some embodiments, the moisture content is less than 4%, less than 3%,less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than0.3%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%,less than 0.075%, less than 0.05%, less than 0.025%, or less than 0.01%.In some preferred embodiments, the moisture content of the formulationsof the present invention is from about 0.01% to about 5%, from about0.01% to about 4%, from about 0.01% to about 3%, from about 0.01% toabout 2%, from about 0.01% to about 1.5%, or from about 0.01% to about1%. In other preferred embodiments, the moisture content of theformulations of the present invention is from about 0.1% to about 5%,from about 0.1% to about 4%, from about 0.1% to about 3%, from about0.1% to about 2%, from about 0.1% to about 1.5%, or from about 0.1% toabout 1%. In other preferred embodiments, the moisture content of theformulations of the present invention is from about 0.25% to about 5%,from about 0.25% to about 4%, from about 0.25% to about 3%, from about0.25% to about 2%, or from about 0.25% to about 1.5%. In still otherpreferred embodiments, the moisture content of the formulations is fromabout 0.5% to about 1%.

E. pH Memory

The “pH memory” of a peptide is the resulting charge profile(protonation state) after drying the peptide from a buffered aqueoussolution (e.g., from a non-volatile buffer). The protonation state, andthus the solubility and stability of peptides, in very low or zeromoisture non-aqueous solvents are affected by the aqueous pH of thepeptide solution before drying and the drying conditions employed. Whenthe peptide is dried in a buffer species in which both the acidic andbasic components are non-volatile, the pH memory of the dried peptidewill be about equal to the pH of the peptide in the non-volatile buffer.See, e.g., Enzymatic Reactions in Organic Media, Koskinen, A. M. P., andKlibanov, A. M., eds., Springer (1996). Furthermore, the pH of thebuffered aqueous solution (e.g., non-volatile buffer) in which thepeptide is dried can be optimized to yield a pH memory for the peptidethat results in optimal peptide stability, maximum solubility, andminimal degradation when the dried peptide is subsequently reconstitutedin an aprotic polar solvent. Because aprotic polar solvents do not haveexchangeable protons, when the dried peptide is reconstituted into anaprotic polar solvent, the reconstituted formulation will maintain thesolubility and stability characteristics of the optimal pH memory.

For stable formulations comprising two, three, four, or more peptides,each peptide is dried so that it has its own pH memory that is optimizedfor maximum solubility, maximum stability, and minimal degradation. Inembodiments where there are two or more peptides in the formulation, thepH memory range of the first peptide may partially overlap with the pHmemory range of the second peptide (e.g., the pH memory of the firstpeptide may be from about 4.0 to about 6.0, and the pH memory of thesecond peptide may be from about 6.0 to about 8.0), or the pH memoryrange of the first peptide may not overlap with the pH memory range ofthe second peptide (e.g., the pH memory of the first peptide may be fromabout 4.0 to about 5.0, and the pH memory of the second peptide may befrom about 6.0 to about 8.0).

The pH memory of a peptide can be measured in several ways. In onemethod, the pH memory of a peptide is measured by reconstituting thedried peptide into un-buffered water and measuring the pH of thereconstituted peptide with a pH indicator such as pH paper or acalibrated pH electrode. Alternatively, the pH memory of a peptide canbe determined for a peptide that has been reconstituted in the aproticpolar solvent (e.g., DMSO) by adding at least 20% water to the aproticpolar solvent (e.g., DMSO) and measuring the pH with a pH indicator.See, e.g., Baughman and Kreevoy, “Determination of Acidity in 80%Dimethyl Sulfoxide-20% Water,” Journal of Physical Chemistry,78(4):421-23 (1974). Measurement of pH in an aprotic polar solvent-watersolution may require a small correction (i.e., no more than 0.2 pH unitas per Baughman and Kreevoy, supra).

In some embodiments, a dried peptide has a pH memory that is about equalto the pH of the peptide in the non-volatile buffer from which it wasdried when the pH memory of the peptide when it is reconstituted in anaprotic polar solvent is within one pH unit of the pH of the peptide inthe non-volatile buffer from which it is dried (thus, for example, for apeptide having a pH of 3.0 in the non-volatile buffer from which thepeptide is dried, a pH memory for the peptide of from 2.0 to 4.0, whenreconstituted in the aprotic polar solvent, would be within one pH unit,and thus the pH memory of the dried peptide would be about equal to thepH of the peptide in the non-volatile buffer). In some embodiments, adried peptide has a pH memory that is about equal to the pH of thepeptide in the non-volatile buffer from which it was dried when the pHmemory of the peptide when it is reconstituted in an aprotic polarsolvent is within half of a pH unit of the pH of the peptide in thenon-volatile buffer from which it is dried (thus, for example, for apeptide having a pH of 3.0 in the non-volatile buffer from which thepeptide is dried, a pH memory for the peptide of from 2.5 to 3.5, whenreconstituted in the aprotic polar solvent, would be within half of a pHunit, and thus the pH memory of the dried peptide would be about equalto the pH of the peptide in the non-volatile buffer).

In some embodiments, the peptide of the stable formulation has a pHmemory of about 1.5 to about 2.5. In some embodiments, the peptide ofthe stable formulation has a pH memory of about 2.0 to about 3.0. Insome embodiments, the peptide of the stable formulation has a pH memoryof about 2.0 to about 4.0. In some embodiments, the peptide of thestable formulation has a pH memory of about 2.5 to about 4.0. In someembodiments, the peptide of the stable formulation has a pH memory ofabout 2.5 to about 3.5. In some embodiments, the peptide of the stableformulation has a pH memory of about 3.0 to about 5.0. In someembodiments, the peptide of the stable formulation has a pH memory ofabout 3.0 to about 4.5. In some embodiments, the peptide of the stableformulation has a pH memory of about 4.0 to about 5.0. In someembodiments, the peptide of the stable formulation has a pH memory ofabout 4.0 to about 6.0. In some embodiments, the peptide of the stableformulation has a pH memory of about 6.0 to about 8.0. In someembodiments, the peptide of the stable formulation has a pH memory ofabout 6.5 to about 8.0. In some embodiments, the peptide of the stableformulation has a pH memory of about 6.5 to about 7.5. In someembodiments, the peptide of the stable formulation has a pH memory ofabout 6.5 to about 9.0. In some embodiments, the peptide of the stableformulation has a pH memory of about 7.0 to about 9.0. In someembodiments, the peptide of the stable formulation has a pH memory ofabout 7.5 to about 9.0. In some embodiments, the peptide of the stableformulation has a pH memory of about 8.0 to about 10.0. In someembodiments, the peptide of the stable formulation has a pH memory ofabout 8.5 to about 10.0. In some embodiments, the pH memory of a peptidemay be about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0,about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0.

F. Exemplary Formulations

In some particular embodiments, the present invention provides a stableglucagon formulation, the glucagon formulation comprising: a glucagonpeptide or salt thereof (e.g., glucagon acetate), wherein the glucagonhas been dried in a non-volatile buffer selected from a glycine buffer,a citrate buffer, a phosphate buffer, and mixtures thereof, and whereinthe dried glucagon has a pH memory that is from about 2.0 to about 3.0;and an aprotic polar solvent selected from the group consisting ofdimethylsulfoxide (DMSO), ethyl acetate, n-methyl pyrrolidone (NMP), andmixtures thereof; wherein the moisture content of the formulation isless than 5%, and wherein the dried glucagon maintains the pH memory ofabout 2.0 to about 3.0 when the dried glucagon is reconstituted in theaprotic polar solvent. In some embodiments, the glucagon is present inthe formulation in an amount ranging from about 0.5 mg/mL to about 100mg/mL, or from about 1 mg/mL to about 50 mg/mL. In some embodiments, themoisture content of the formulation is less than about 2%, less thanabout 1%, less than about 0.5%, or less than about 0.01%. In someembodiments, the moisture content of the formulation is from about 0.01%to about 3%. In some embodiments, the formulation further comprises astabilizing excipient selected from sugars (e.g., trehalose), starches(e.g., hydroxyethyl starch (HES)), and mixtures thereof. The stabilizingexcipient may be present in the formulation in an amount ranging fromabout 1% (w/v) to about 60% (w/v). In some embodiments, the formulationfurther comprises a co-solvent that depresses the freezing point of theformulation, wherein the co-solvent is selected from ethanol, propyleneglycol, glycerol, and mixtures thereof. The co-solvent may be present inthe formulation in an amount ranging from about 10% (w/v) to about 50%(w/v).

In other particular embodiments, the present invention provides a stableglucagon formulation, the glucagon formulation comprising: a glucagonpeptide or salt thereof (or glucagon analog or peptidomimetic); and anaprotic polar solvent selected from the group consisting ofdimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP) and mixturesthereof; wherein the moisture content of the formulation is less than3%. In preferred embodiments, the moisture content of the formulation isless than 2%, less than 1%, less than 0.5% and less than 0.25%. In otherpreferred embodiments, the moisture content is from 0.25% to about 3%,preferably from about 0.25% to about 2%, more preferably from about0.25% to about 1.5%, more preferably from about 0.25% to about 1%, morepreferably from about 0.5% to about 1%.

In other particular embodiments, the stable glucagon formulation furthercomprises a non-volatile buffer and a stabilizing excipient that is asugar, a starch, or a sugar alcohol. For instance, in some embodiments,the glucagon formulation further comprises a glycine buffer andmannitol, or a citrate buffer and mannitol, or a phosphate buffer andmannitol. In some embodiments, the glucagon formulation furthercomprises a glycine buffer and trehalose, or a citrate buffer andtrehalose, or a phosphate buffer and trehalose. In these embodiments,the aprotic polar solvent can be DMSO, NMP, ethyl acetate, or a mixturethereof. For instance, in one preferred embodiment, the aprotic polarsolvent is DMSO, and the non-volatile buffer is a glycine buffer. Inanother preferred embodiment, the aprotic polar solvent is DMSO, thenon-volatile buffer is a citrate buffer and the stabilizing excipient ismannitol. In another preferred embodiments, the aprotic polar solvent isDMSO, the non-volatile buffer is a glycine buffer, and the stabilizingexcipient is trehalose. In still another preferred embodiment, theaprotic polar solvent is DMSO, and the non-volatile buffer is a citratebuffer. In still another preferred embodiment, the aprotic polar solventis NMP, and the non-volatile buffer is a glycine buffer.

In other particular embodiments, the present invention provides a stableformulation comprising: glucagon or a salt thereof (e.g., glucagonacetate), wherein the glucagon has been dried in a non-volatile buffer,and wherein the dried glucagon has a pH memory that is about equal tothe pH of the glucagon in the non-volatile buffer selected from aglycine buffer, a citrate buffer, a phosphate buffer, and mixturesthereof, wherein the pH memory of the dried glucagon is from about 2.0to about 3.0; and an aprotic polar solvent selected fromdimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, andmixtures thereof; wherein the moisture content of the formulation isless than 1%, and wherein the dried glucagon maintains the pH memorythat is about equal to the pH of the glucagon in the non-volatile bufferwhen the dried glucagon is reconstituted in the aprotic polar solvent.In some embodiments, the glucagon formulation further comprises aco-solvent that depresses the freezing point of the formulation, whereinthe co-solvent is selected from ethanol, propylene glycol, glycerol, andmixtures thereof. In some embodiments, the glucagon formulation furthercomprises a stabilizing excipient selected from sugars, starches, andmixtures thereof. In some embodiments, the glucagon is present in theformulation in an amount ranging from about 1 mg/mL to about 50 mg/mL.

In other particular embodiments, the present invention provides a stableglucagon formulation, the glucagon formulation consisting essentiallyof: a glucagon peptide or salt thereof (e.g., glucagon acetate), whereinthe glucagon has been dried in a non-volatile buffer selected from aglycine buffer, a citrate buffer, a phosphate buffer, and mixturesthereof, and wherein the dried glucagon has a pH memory that is fromabout 2.0 to about 3.0; and an aprotic polar solvent selected from thegroup consisting of dimethylsulfoxide (DMSO), ethyl acetate, n-methylpyrrolidone (NMP), and mixtures thereof; wherein the moisture content ofthe formulation is less than 5%, and wherein the dried glucagonmaintains the pH memory of about 2.0 to about 3.0 when the driedglucagon is reconstituted in the aprotic polar solvent.

In still other particular embodiments, the present invention provides astable glucagon formulation, the glucagon formulation consistingessentially of: a glucagon peptide or salt thereof (e.g., glucagonacetate), wherein the glucagon has been dried in a non-volatile bufferselected from a glycine buffer, a citrate buffer, a phosphate buffer,and mixtures thereof, and wherein the dried glucagon has a pH memorythat is from about 2.0 to about 3.0; and a mixture of an aprotic polarsolvent and a co-solvent that depresses the freezing point of theformulation, wherein the aprotic polar solvent is selected from thegroup consisting of dimethylsulfoxide (DMSO), ethyl acetate, n-methylpyrrolidone (NMP), and mixtures thereof and wherein the co-solvent isselected from ethanol, propylene glycol, glycerol, and mixtures thereof;wherein the moisture content of the formulation is less than 5%, andwherein the dried glucagon maintains the pH memory of about 2.0 to about3.0 when the dried glucagon is reconstituted in the aprotic polarsolvent.

In other particular embodiments, the present invention provides a stableglucagon formulation, the glucagon formulation consisting essentiallyof: a glucagon peptide or salt thereof (e.g., glucagon acetate), whereinthe glucagon has been dried in a mixture of a non-volatile buffer and astabilizing excipient, wherein the non-volatile buffer is selected froma glycine buffer, a citrate buffer, a phosphate buffer, and mixturesthereof, and the stabilizing excipient is selected from sugars (e.g.,trehalose), starches (e.g., hydroxyethyl starch (HES)), and mixturesthereof, and wherein the dried glucagon has a pH memory that is fromabout 2.0 to about 3.0; and an aprotic polar solvent selected from thegroup consisting of dimethylsulfoxide (DMSO), ethyl acetate, n-methylpyrrolidone (NMP), and mixtures thereof; wherein the moisture content ofthe formulation is less than 5%, and wherein the dried glucagonmaintains the pH memory of about 2.0 to about 3.0 when the driedglucagon is reconstituted in the aprotic polar solvent.

In still other particular embodiments, the present invention provides astable formulation comprising: insulin, wherein the insulin has beendried in a first non-volatile buffer selected from a glycine buffer, acitrate buffer, a phosphate buffer, and mixtures thereof, and whereinthe dried insulin has a first pH memory that is about equal to the pH ofthe insulin in the first non-volatile buffer, wherein the first pHmemory is from about 1.5 to about 2.5, or from about 6.0 to about 8.0;pramlintide, wherein the pramlintide has been dried in a secondnon-volatile buffer selected from a glycine buffer, a citrate buffer, aphosphate buffer, and mixtures thereof, and wherein the driedpramlintide has a second pH memory that is about equal to the pH of thepramlintide in the second non-volatile buffer, wherein the second pHmemory is from about 3.0 to about 5.0, or from about 4.0 to about 6.0;and an aprotic polar solvent selected from dimethylsulfoxide (DMSO),n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof; whereinthe moisture content of the formulation is less than 1%, wherein thedried insulin maintains the first pH memory that is about equal to thepH of the insulin in the first non-volatile buffer when the driedinsulin is reconstituted in the aprotic polar solvent, and wherein thedried pramlintide maintains the second pH memory that is about equal tothe pH of the pramlintide in the second non-volatile buffer when thedried pramlintide is reconstituted in the aprotic polar solvent. In someembodiments, the insulin and pramlintide formulation further comprises aco-solvent that depresses the freezing point of the formulation, whereinthe co-solvent is selected from ethanol, propylene glycol, glycerol, andmixtures thereof. In some embodiments, one or both of the insulin in thefirst non-volatile buffer and the pramlintide in the second non-volatilebuffer further comprises a stabilizing excipient selected from sugars,starches, and mixtures thereof. In some embodiments, the firstnon-volatile buffer and the second non-volatile buffer are the same. Insome embodiments, the first non-volatile buffer and the secondnon-volatile buffer are different. In some embodiments, each of theinsulin and pramlintide is present in the formulation in an amountranging from about 1 mg/mL to about 50 mg/mL. In some embodiments, thefirst pH memory is from about 1.5 to about 2.5. In some embodiments, thefirst pH memory is from about 6.0 to about 8.0. In some embodiments, thesecond pH memory is from about 3.0 to about 5.0. In some embodiments,the second pH memory is from about 4.0 to about 6.0. In someembodiments, the first pH memory is from about 1.5 to about 2.5 and thesecond pH memory is from about 3.0 to about 5.0.

IV. Methods of Making Stable Peptide Formulations

In yet another aspect, the present invention provides a process formaking a stable formulation for parenteral injection. In someembodiments, the process comprises: drying a peptide and a non-volatilebuffer to a dry peptide powder; and reconstituting the dried peptidepowder with an aprotic polar solvent, thereby making the stableformulation, wherein the moisture content of the stable formulation isless than 5%. In some embodiments, the dried peptide powder has a pHmemory that is about equal to the pH of the peptide in the non-volatilebuffer, and the dried peptide powder maintains the pH memory that isabout equal to the pH of the peptide in the non-volatile buffer when thedried peptide powder is reconstituted in the aprotic polar solvent.

The process for making stable peptide formulations can be used toformulate any peptide that has limited or poor stability or solubilityin an aqueous environment. Peptides (or salts thereof) suitable for usein the formulations of the present invention include, but are notlimited to, glucagon, insulin, leuprolide, anluteinizing-hormone-releasing hormone (LHRH) agonists, pramlintide,parathyroid hormone (PTH), amylin, botulinum toxin, a conotoxin,hematide, an amyloid peptide, cholecystikinin, gastric inhibitorypeptide, an insulin-like growth factor, growth hormone releasing factor,anti-microbial factor, glatiramer, glucagon-like peptide-1 (GLP-1), aGLP-1 agonist, exenatide, and analogs thereof. In a preferredembodiment, the peptide is glucagon or a glucagon analog orpeptidomimetic. In another embodiment, the peptide is parathyroidhormone. In yet another embodiment, the peptide is leuprolide. In stillanother embodiment, the peptide is glatiramer.

In some embodiments, two, three, four or more peptides are formulatedinto a stable formulation. In embodiments where two or more peptides areformulated into the stable formulation, each peptide is separately driedwith a non-volatile buffer to a dry peptide powder, and each driedpeptide powder has a pH memory that is about equal to the pH of thepeptide in the non-volatile buffer (i.e., the first peptide has a firstpH memory that is about equal to the pH of the first peptide in thefirst non-volatile buffer, and the second peptide has a second pH memorythat is about equal to the pH of the second peptide in the secondnon-volatile buffer). The two or more dried peptide powders arereconstituted with an aprotic polar solvent, thereby making the stableformulation, wherein the moisture content of the stable formulation isless than 5%, and wherein each dried peptide powder maintains the pHmemory that is about equal to the pH of the peptide in the non-volatilebuffer when the dried peptide powder is reconstituted in the aproticpolar solvent (i.e., the first dried peptide maintains the first pHmemory when the first dried peptide is reconstituted in the aproticpolar solvent, and the second dried peptide maintains the second pHmemory when the second dried peptide is reconstituted in the aproticpolar solvent).

In the process for making stable peptide formulations, suitablenon-volatile buffers include, for example, glycine buffers, citratebuffers, phosphate buffers, and mixtures thereof. In some embodiments,the non-volatile buffer is a glycine buffer or a citrate buffer. In someembodiments, the non-volatile buffer is a mixture of a citrate bufferand a phosphate buffer. In some embodiments, the peptide is mixed withboth the non-volatile buffer and a stabilizing excipient (such as asugar, a starch, or mixtures thereof) and then dried to a dried peptidepowder. In other embodiments, the stabilizing excipient (such as asugar, a starch, a sugar alcohol, or mixtures thereof) is added to thereconstituted peptide in the aprotic polar solvent. In some embodiments,the stabilizing excipient is present in the formulation in an amountranging from about 1% (w/v) to about 60% (w/v), e.g., about 1%, about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, or about 60% (w/v). In someembodiments, the stabilizing excipient is trehalose. In someembodiments, the stabilizing excipient is HES. In some embodiments, thestabilizing excipient is a mixture of trehalose and HES.

As explained above, when the peptide is mixed with the non-volatilebuffer, the non-volatile buffer is selected such that the peptide has apH of maximal stability/minimal degradation in the aqueous environment.Once dried, the peptide will have a pH memory of maximalstability/minimal degradation and will retain that pH memory whendissolved in or reconstituted in the aprotic polar solvent. As such, inone embodiment, the pH of the non-volatile buffer is such that the driedpeptide powder has a pH memory of about 2 to about 3. In anotherembodiment, the pH of the non-volatile buffer is such that the driedpeptide powder has a pH memory of about 4 to about 6. In yet anotherembodiment, the pH of the non-volatile buffer is such that the driedpeptide powder has a pH memory of about 4 to about 5. In yet anotherembodiment, the pH of the non-volatile buffer is such that the driedpeptide powder has a pH memory of about 6 to about 8.

Once the peptide and the non-volatile buffer (and optionally othercomponents, such as a stabilizing excipient, that are added to thepeptide and the non-volatile buffer before drying) are dried to apowder, the dried peptide powder is dissolved or reconstituted in anaprotic polar solvent as described herein (e.g., dimethylsulfoxide(DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and mixturesthereof). In some embodiments, the aprotic polar solvent isdimethylsulfoxide (DMSO). In other embodiments, the aprotic polarsolvent is n-methyl pyrrolidone (NMP).

In some embodiments, the step of reconstituting the dried peptide powdercomprises diluting or reconstituting the dried peptide with a mixturecomprising an aprotic polar solvent and a co-solvent that depresses thefreezing point of the formulation. In some embodiments, the co-solventis selected from ethanol, propylene glycol, glycerol, and mixturesthereof. In some embodiments, the co-solvent is present in theformulation in an amount ranging from about 10% (w/v) to about 50%(w/v), e.g., about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, or about 50% (w/v).

The formulations of the present invention have very little residualmoisture and, thus, the peptides in such formulations remain stable overextended periods of time. In preferred embodiments, the moisture contentof the stable formulation that is made by the process of the presentinvention is less than 4%, less than 3%, less than 2%, less than 1%,less than 0.5%, less than 0.4%, less than 0.3%, less than 0.25%, lessthan 0.2%, less than 0.15%, less than 0.1%, less than 0.075%, less than0.05%, less than 0.025%, or less than 0.01%.

In the foregoing process, drying of the peptide compound with thenon-volatile buffer (and optionally the stabilizing excipient) iscarried out using spray-drying techniques, freeze-drying techniques orlyophilization techniques. Spray-drying techniques are well known tothose skilled in the art. Spray-drying includes the steps of atomizationof a solution containing one or more solid (e.g., therapeutic agent) viaa nozzle spinning disk, or other device, followed by evaporation of thesolvent from the droplets. The nature of the powder that results is thefunction of several variables including the initial soluteconcentration, size distribution of droplets produced and the rate ofsolute removal. The particles produced may comprise aggregates ofprimary particles which consist of crystals and/or amorphous solidsdepending on the rate and conditions of solvent removal.

A spray-drying process for preparing ultra-fine powders of biologicalmacromolecules such as proteins, oligopeptides, high molecular weightpolysaccharides, and nucleic acids is described in, for example, U.S.Pat. No. 6,051,256. Freeze-drying procedures are well known in the art,and are described, for example, in U.S. Pat. No. 4,608,764 and U.S. Pat.No. 4,848,094. Spray-freeze-drying processes are described, e.g., inU.S. Pat. No. 5,208,998. Other spray-drying techniques are described,for example, in U.S. Pat. Nos. 6,253,463; 6,001,336; 5,260,306; and PCTInternational Publication Nos. WO 91/16882 and WO 96/09814.

Lyophilization techniques are well known to those skilled in the art.Lyophilization is a dehydration technique that takes place while aproduct is in a frozen state (ice sublimation under a vacuum) and undera vacuum (drying by gentle heating). These conditions stabilize theproduct, and minimize oxidation and other degradative processes. Theconditions of freeze drying permit running the process at lowtemperatures, therefore, thermally labile products can be preserved.Steps in freeze drying include pretreatment, freezing, primary dryingand secondary drying. Pretreatment includes any method of treating theproduct prior to freezing. This may include concentrating the product,formulation revision (i.e., addition of components to increase stabilityand/or improve processing), decreasing a high vapor pressure solvent orincreasing the surface area. Methods of pretreatment include: freezeconcentration, solution phase concentration, and formulatingspecifically to preserve product appearance or to provide lyoprotectionfor reactive products, and are described, e.g., in U.S. Pat. No.6,199,297. “Standard” lyophilization conditions, are described, e.g., inU.S. Pat. No. 5,031,336, and in “Freeze Drying of Pharmaceuticals”(DeLuca, Patrick P., J. Vac. Sci. Technol., Vol. 14, No. 1,January/February 1977); and “The Lyophilization of Pharmaceuticals: ALiterature Review” (Williams, N. A., and G. P. Polli, Journal ofParenteral Science and Technology, Vol. 38, No. 2, March/April 1984).

In certain preferred embodiments, the lyophilization cycle is partiallyperformed above the glass transition temperature (Tg) of the therapeuticagent formulation to induce a collapse of the mass to form a dense cakecontaining residue moisture. In other embodiments, the lyophilizationcycle is carried out below the glass transition temperature in order toavoid a collapse in order to achieve a complete drying of the particles.

V. Therapeutic Methods

In another aspect, the present invention provides methods of treatingdiseases or conditions by administering to a subject a stableformulation as described herein in an amount effective to treat,alleviate or prevent the disease, condition or disorder. In someembodiments, the disease, condition, or disorder to be treated with astable formulation of the present invention is a diabetic condition.Examples of diabetic conditions include, but are not limited to, type 1diabetes, type 2 diabetes, gestational diabetes, pre-diabetes,hyperglycemia, hypoglycemia, and metabolic syndrome. In someembodiments, the disease, condition, or disorder is hypoglycemia. Insome embodiments, the disease, condition, or disorder is diabetes.

In some embodiments, a therapeutic method of the present inventioncomprises treating hypoglycemia by administering to a subject havinghypoglycemia a stable formulation as described herein in an amounteffective to treat the hypoglycemia. In some embodiments, the subject isadministered a stable formulation comprising glucagon.

In some embodiments, a therapeutic method of the present inventioncomprises treating diabetes by administering to a subject havingdiabetes a stable formulation as described herein in an amount effectiveto treat the diabetes. In some embodiments, the subject is administereda stable formulation comprising insulin. In some embodiments, thesubject is administered a stable formulation comprising pramlintide. Insome embodiments, the subject is administered a stable formulationcomprising insulin and pramlintide. In some embodiments, the subject isadministered a stable formulation comprising exenatide. In someembodiments, the subject is administered a stable formulation comprisingglucagon and exenatide.

Administered dosages for the peptide drugs as described herein fortreating a disease, condition, disorder (e.g., a diabetic condition,e.g., hypoglycemia or diabetes) are in accordance with dosages andscheduling regimens practiced by those of skill in the art. Generalguidance for appropriate dosages of all pharmacological agents used inthe present methods is provided in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 11th Edition, 2006, supra, and ina Physicians' Desk Reference (PDR), for example, in the 65th (2011) or66th (2012) Eds., PDR Network, LLC, each of which is hereby incorporatedherein by reference. The appropriate dosage of a peptide drug fortreating a disease, condition, or disorder as described herein will varyaccording to several factors, including the formulation of thecomposition, patient response, the severity of the condition, thesubject's weight, and the judgment of the prescribing physician.Effective doses of the described formulations deliver a medicallyeffective amount of a peptide drug. The dosage can be increased ordecreased over time, as required by an individual patient.

Determination of an effective amount or dose is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. Generally, the formulations todeliver these doses may contain one, two, three, four, or more peptidesor peptide analogs (collectively “peptide,” unless peptide analogs areexpressly excluded), wherein each peptide is present at a concentrationfrom about 0.1 mg/mL up to the solubility limit of the peptide in theformulation. This concentration is preferably from about 1 mg/mL toabout 100 mg/mL, e.g., about 1 mg/mL, about 5 mg/mL, about 10 mg/mL,about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL,about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, or about 100mg/mL.

The formulations of the present invention may be for subcutaneous,intradermal, or intramuscular administration (e.g., by injection or byinfusion). In some embodiments, the formulation is administeredsubcutaneously.

The formulations of the present disclosure are administered by infusionor by injection using any suitable device. For example, a formulation ofthe present invention may be placed into a syringe, a pen injectiondevice, an auto-injector device, or a pump device. In some embodiments,the injection device is a multi-dose injector pump device or amulti-dose auto-injector device. The formulation is presented in thedevice in such a fashion that the formulation is readily able to flowout of the needle upon actuation of an injection device, such as anauto-injector, in order to deliver the peptide drugs. Suitablepen/autoinjector devices include, but are not limited to, thosepen/autoinjection devices manufactured by Becton-Dickenson, SwedishHealthcare Limited (SHL Group), YpsoMed Ag, and the like. Suitable pumpdevices include, but are not limited to, those pump devices manufacturedby Tandem Diabetes Care, Inc., Delsys Pharmaceuticals and the like.

In some embodiments, the formulations of the present invention areprovided ready for administration in a vial, a cartridge, or apre-filled syringe.

In another aspect, the present invention provides for the use of astable formulation as described herein for the formulation of amedicament for the treatment of any disease, condition, or disorder thatmay be treated with the peptide of the formulation. In some embodiments,the stable formulation is used for formulating a medicament for thetreatment of a diabetic condition, e.g., type 1 diabetes, type 2diabetes, gestational diabetes, pre-diabetes, hyperglycemia,hypoglycemia, or metabolic syndrome.

In some embodiments, the stable formulation is used for formulating amedicament for the treatment of hypoglycemia. In some embodiments, thestable formulation comprises glucagon or a salt thereof (e.g., glucagonacetate). In some embodiments, the stable formulation comprises glucagonand exenatide.

In some embodiments, the stable formulation is used for formulating amedicament for the treatment of diabetes. In some embodiments, thestable formulation comprises insulin. In some embodiments, the stableformulation comprises exenatide. In some embodiments, the stableformulation comprises pramlintide. In some embodiments, the stableformulation comprises insulin and pramlintide.

VI. Kits

In another aspect, the present invention kits for treating a disease,condition or disorder as described herein. In some embodiments, the kitcomprises: a stable formulation comprising one, two, three, four or morepeptides or salts thereof, wherein the peptide(s) has been dried in anon-volatile buffer, and wherein the dried peptide(s) has a pH memorythat is about equal to the pH of the peptide(s) in the non-volatilebuffer; and an aprotic polar solvent; wherein the moisture content ofthe formulation is less than 5%, and wherein the dried peptide(s)maintains the pH memory that is about equal to the pH of the peptide(s)in the non-volatile buffer when the dried peptide(s) is reconstituted inthe aprotic polar solvent; and a syringe for administration of thestable formulation to the subject.

In some embodiments, the kit comprises a stable glucagon formulation asdescribed herein for use in treating hypoglycemia in a subject. In someembodiments, the kit comprises a glucagon formulation comprising:glucagon or a salt thereof (e.g., glucagon acetate), wherein theglucagon has been dried in a non-volatile buffer, and wherein the driedglucagon has a pH memory that is about equal to the pH of the glucagonin the non-volatile buffer selected from a glycine buffer, a citratebuffer, a phosphate buffer, and mixtures thereof, wherein the pH memoryof the dried glucagon is from about 2.0 to about 3.0; and an aproticpolar solvent selected from dimethylsulfoxide (DMSO), n-methylpyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein themoisture content of the formulation is less than 1%, and wherein thedried glucagon maintains the pH memory that is about equal to the pH ofthe glucagon in the non-volatile buffer when the dried glucagon isreconstituted in the aprotic polar solvent. In some embodiments, theglucagon formulation further comprises a co-solvent that depresses thefreezing point of the formulation, wherein the co-solvent is selectedfrom ethanol, propylene glycol, glycerol, and mixtures thereof. In someembodiments, the glucagon formulation further comprises a stabilizingexcipient selected from sugars, starches, and mixtures thereof. In someembodiments, the glucagon is present in the formulation in an amountranging from about 1 mg/mL to about 50 mg/mL.

In some embodiments, the kit comprises a stable insulin and pramlintideformulation as described herein for use in treating diabetes in asubject. In some embodiments, the kit comprises an insulin andpramlintide formulation comprising: insulin, wherein the insulin hasbeen dried in a first non-volatile buffer selected from a glycinebuffer, a citrate buffer, a phosphate buffer, and mixtures thereof, andwherein the dried insulin has a first pH memory that is about equal tothe pH of the insulin in the first non-volatile buffer, wherein thefirst pH memory is from about 1.5 to about 2.5 or from about 6.0 toabout 8.0; pramlintide, wherein the pramlintide has been dried in asecond non-volatile buffer selected from a glycine buffer, a citratebuffer, a phosphate buffer, and mixtures thereof, and wherein the driedpramlintide has a second pH memory that is about equal to the pH of thepramlintide in the second non-volatile buffer, wherein the second pHmemory is from about 3.0 to about 5.0 or from about 4.0 to about 6.0;and an aprotic polar solvent selected from dimethylsulfoxide (DMSO),n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof; whereinthe moisture content of the formulation is less than 1%, wherein thedried insulin maintains the first pH memory that is about equal to thepH of the insulin in the first non-volatile buffer when the driedinsulin is reconstituted in the aprotic polar solvent, and wherein thedried pramlintide maintains the second pH memory that is about equal tothe pH of the pramlintide in the second non-volatile buffer when thedried pramlintide is reconstituted in the aprotic polar solvent. In someembodiments, the insulin and pramlintide formulation further comprises aco-solvent that depresses the freezing point of the formulation, whereinthe co-solvent is selected from ethanol, propylene glycol, glycerol, andmixtures thereof. In some embodiments, one or both of the insulin in thefirst non-volatile buffer and the pramlintide in the second non-volatilebuffer further comprises a stabilizing excipient selected from sugars,starches, and mixtures thereof. In some embodiments, the firstnon-volatile buffer and the second non-volatile buffer are the same. Insome embodiments, the first non-volatile buffer and the secondnon-volatile buffer are different. In some embodiments, each of theinsulin and pramlintide is present in the formulation in an amountranging from about 1 mg/mL to about 50 mg/mL. In some embodiments, thefirst pH memory is from about 1.5 to about 2.5. In some embodiments, thefirst pH memory is from about 6.0 to about 8.0. In some embodiments, thesecond pH memory is from about 3.0 to about 5.0. In some embodiments,the second pH memory is from about 4.0 to about 6.0. In someembodiments, the first pH memory is from about 1.5 to about 2.5 and thesecond pH memory is from about 3.0 to about 5.0.

In some embodiments, the kit comprises a syringe that is part of a peninjection device, an auto-injector device or a pump. In some embodiment,the syringe is prefilled with the stable formulation. In someembodiments, the kit further comprises instructions, wherein theinstructions direct the administration of the stable formulation totreat the subject in need thereof (e.g., the subject having hypoglycemiaor diabetes).

VII. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Preparation of Glucagon Solutions for Use in Freeze-Drying

Various solutions were prepared to contain glucagon at a concentrationof 10 mg/mL. The solutions contained, alternatively, glycine, citrate orphosphate at 5 mM, generally providing a buffer establishing a pH of 3.The solution also contained a sugar, alone or in combination, in amountsequal to the w/v amount of glucagon (1:1) or at 200% (2:1) of the amountof glucagon. The sugars were trehalose, HES, and β-cyclodextrin (β-CD).Some solutions also contained Tween-20 at 0.10% w/v as a surfactant. Thevarious formulations were mixed to substantial homogeneity in amounts asdescribed in Table 1 below.

TABLE 1 Glucagon Mixtures for Subsequent Lyophilization Glycine CitratePhosphate Formulation Glucagon Buffer Buffer Buffer Trehalose HES β-CDTween-20 # (mg/ml) (mM) (mM) (mM) (mg/ml) (mg/ml) (mg/ml) (mg/ml) 1 5 50 0 0 0 0 0 2 5 5 0 0 0 0 0 0.01 3 5 5 0 0 10 0 0 0 4 5 5 0 0 0 10 0 0 55 5 0 0 5 5 0 0 6 5 5 0 0 0 0 10 0 7 5 0 5 0 0 0 0 0 8 5 0 5 0 0 0 00.01 9 5 0 5 0 10 0 0 0 10 5 0 5 0 0 10 0 0 11 5 0 5 0 5 5 0 0 12 5 0 50 0 0 10 0 13 5 0 0 5 0 0 0 0 14 5 0 0 5 0 0 0 0.01 15 5 0 0 5 10 0 0 016 5 0 0 5 0 10 0 0 17 5 0 0 5 5 5 0 0 18 5 0 0 5 0 0 10 0 19 5 5 0 0 100 0 0.01 20 5 5 0 0 0 10 0 0.01 21 5 5 0 0 5 5 0 0.01

To prepare the mixtures, the glucagon was dissolved in the respectivebuffers (phosphate, citrate, and/or glycine buffers, 5 mM, pH 3.0) at 10mg/mL. The solution was then mixed in a 1:1 (v/v) ratio with varioussolutes, which were prepared at twice the desired concentration usingcorresponding buffer, in order to obtain a final glucagon concentrationof 5 mg/mL and the final desired solute concentration. The solutionswere then filtered through 0.2 μm Millipore PES membrane to removeinsoluble materials. The sample preparations were conducted in a 4° C.cold room. The glucagon concentration and the purity were determined byRP- and Size-Exclusion (SE)-HPLC.

Example 2 Preparation of Dry Glucagon Powder by Freeze-Drying

The above formulations of Table 1 were pipetted (0.3 mL) into 3-mLlyophilization vials (13-mm ID). The formulations were lyophilized in aFTS Durastop freeze-drier (Stoneridge, N.Y.). Samples were frozen at−40° C. at a ramp of 2.5° C./min and maintained for 2 hours (h) to allowsufficient freezing. The sample temperature was then increased to −5° C.at a ramp of 2° C./min and held for 2 h as an annealing step. Thetemperature was then decreased to 30° C. at a ramp of 1.5° C./min andthe vacuum was turned on at 60 mTorr. The primary drying was set for 24h. The temperature was gradually increased to 40° C. at a ramp of 0.5°C./min and held for additional 10 h. After drying was complete, thevials were capped under vacuum using XX stoppers from the WestPharmaceutical company (product #10123524). None of the formulationsshowed any evidence of cake collapse following freeze-drying. Themoisture content of the final dried product was less than 1% w/w.

Example 3 Preparation of Glucagon Formulations in Aprotic Polar Solvents

Six of the dry powders made from the solutions in Table 1 were selectedfor formulation in polar, aprotic solvents:

-   -   1. Buffer (glycine)+trehalose (200% relative to glucagon)        (formulation #3)    -   2. Buffer (glycine)+HES (200% relative to glucagon) (formulation        #4)    -   3. Buffer (glycine)+trehalose (100% relative to glucagon)+HES        (100% relative to glucagon) (formulation #5)    -   4. Buffer (glycine)+Tween-20 (0.01% w/v)+trehalose (200%        relative to glucagon) (formulation #19)    -   5. Buffer (glycine)+Tween-20 (0.01% w/v)+HES (200% relative to        glucagon) (formulation #20)    -   6. Buffer (glycine)+Tween-20 (0.01% w/v)+trehalose (100%        relative to glucagon)+HES (100% relative to glucagon)        (formulation #21)

Example 4 Preparation of a Glucagon Solution with a pH Memory of 4-5

Solutions were prepared to contain glucagon at a concentration of 10-20mg/mL. The solutions contained a citrate buffer establishing pH of 4-5.The solution also contained a sugar alcohol, mannitol, at aconcentration of 50-100 mg/mL. The formulation was mixed to substantialhomogeneity and freeze-dried via the drying cycle described in Example 2to a residual moisture of less than 0.5% w/w. The dry powder isdissolved into DMSO to a concentration of 10-20 mg/mL of glucagon and50-100 mg/mL of mannitol.

Example 5 Preparation of a PTH(1-34) Solution with Low Moisture and LowFreezing Point

Solutions were prepared to contain PTH (1-34) at a concentration of10-20 mg/mL. The solutions contained a citrate buffer establishing pH of4-5. The solution also contained a sugar alcohol, mannitol, at aconcentration of 50 mg/mL. The formulation was mixed to substantialhomogeneity and freeze-dried via the drying cycle described in Example 2to a residual moisture of less than 0.5% w/w. The dry powder isdissolved into DMSO to a concentration of 10-20 mg/mL of PTH (1-34) and50-100 mg/mL of mannitol.

Example 6 Increase in Both Blood Glucagon and Blood Glucose LevelsFollowing Administration of Glucagon Formulation

Two nonaqueous glucagon formulations in aprotic polar solvents, based onglucagon-glycine-trehalose powders dissolved in NMP or DMSO, were testedin a rat pharmacokinetic and pharmacodynamic study and compared with anaqueous formulation. Rats were all dosed at a rate of 10 μgglucagon/rat. The nonaqueous glucagon solutions were given as 10 μLsubcutaneous injections, as was the aqueous control solution. Allformulations tested demonstrated a rapid rise in blood glucagonconcentrations (see FIG. 1).

Pharmacokinetic (PK) parameters were analyzed for the four treatmentgroups plus the aqueous control. A noncompartmental PK analysis wasperformed for each rat. C_(max) and T_(max) were computed from observeddata. Area-under-the-curve (AUC) estimates were computed withoutextrapolation. Data were analyzed using a five group ANOVA to compare PKparameters across groups. No significant differences in either C_(max),T_(max)), or AUC among the three groups was observed. The relativebioavailabilities of the NMP and DMSO formulations relative to theaqueous control group were all close to 100% (76% and 92%,respectively). Thus, the nonaqueous formulations are essentiallybioequivalent to the aqueous glucagon formulation based on the resultsof these rat PK studies.

As predicted from the pharmacokinetic results, the nonaqueous glucagonformulations produced pharmacodynamic profiles essentially equivalent toan aqueous-reconstituted glucagon formulation at the same dose level(see, FIG. 2).

Example 7 Enhanced Solubility of Glucagon in Aprotic Polar SolventsCompared to Aqueous Solutions

Glucagon was prepared at 1.0 mg/mL via dissolution in one of thefollowing buffers:

1. 2 mM citric acid, pH 2.0 (titrated with concentrated HCl) (“C2.0”)

2. 2 mM citric acid, pH 3.0 (titrated with concentrated HCl) (“C3.0”)

Each formulation was placed in sterile 2 cc vials, at 1 mL fill volume.Samples were freeze-dried to low residual moisture and reconstituted tovarious nominal concentrations in DMSO, NMP, or a 50/50 DMSO/NMPco-solvent. Reconstitution concentrations ranged from 1 to 30 mg/mL.Solubility was measured by visual inspection for clarity, turbidity viaA₆₃₀, and RP-HPLC.

As shown in Table 2 below, glucagon lyophilized with a citrate buffer atpH memories of 2.0 and 3.0 were readily soluble to concentrations of 30mg/mL. The same formulations were only fully soluble in H₂O at lowerconcentrations. For pH memory of 3.0, complete reconstitution was onlyachieved at 5 mg/mL in H₂O. Further, glucagon solubilized in H₂O wasonly meta-stable, i.e., it only remained soluble for a few hours andthen began to gel or fibrillate with rates dependent on pH andconcentration, whereas glucagon solubilized in the aprotic polarsolvents/co-solvents were stable indefinitely.

TABLE 2 Solubility of glucagon at pH memory of 2.0 and 3.0 Formula- 1 510 20 30 tion Solvent mg/ml mg/ml mg/ml mg/ml mg/ml C2.0 H₂0 1 5 10 1824 DMSO 1 5 10 20 30 DMSO/NMP 1 5 10 20 30 NMP 1 5 10 20 30 C3.0 H₂0 1 57 17 9 DMSO 1 5 10 20 30 DMSO/NMP 1 5 10 20 30 NMP 1 5 10 20 30

Example 8 Effect of pH on the Solubility of Glucagon in Aprotic PolarSolvents

When the data shown in Example 8 and Table 2 is viewed from a pH memoryperspective, it is apparent that higher solubilities for glucagon can beachieved in the aprotic polar solvents at a lower pH memory (e.g., pH2.0) than at a higher pH. Furthermore, although the recoveries in Table2 indicate essentially 100% of the nominal concentration, A₆₃₀measurements showed increasing turbidity of 30 mg/mL solutions ofglucagon at pH memory of 3.0 (C3.0) in neat NMP and the DMSO/NMPco-solvent, whereas the C2.0 formulations with a pH memory of 2.0remained essentially free of turbidity.

In another example, the effect of pH on the solubility of glucagon inaprotic polar solvents was measured for glucagon acetate dissolved inH₂O at 2 mg/mL with either 2 mL glycine or 2 mM citrate buffer and pHadjusted to the desired value. Samples were freeze-dried andreconstituted to various nominal concentrations in DMSO, NMP, or a 50/50DMSO/NMP co-solvent. Solubility was measured by visual inspection forclarity, turbidity via A₆₃₀, and RP-HPLC.

It was found that “pH memory” from lyophilization had a major effect onglucagon stability. Glucagon was soluble at up to 30 mg/mLreconstitution for “G2.5” (pH memory 2.5) lyophiles DMSO, DMSO/NMP, andNMP. Significantly reduced solubility was observed for “G3.5” (pH memory3.5) lyophiles. G3.5 lyophiles all were cloudy and recoveries were lessthan complete, even at a nominal reconstitution concentration of 10mg/mL. DMSO and the DMSO/NMP co-solvent showed about 95% recovery,whereas NMP showed only about 60% recovery.

Example 9 Effect of Buffer Species on Glucagon Stability in DMSO

Glucagon acetate was prepared at 1.0 mg/mL via dissolution in one of thefollowing buffers:

1. 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl)

2. 2 mM citric acid, pH 3.0 (titrated with concentrated HCl)

These formulations were lyophilized and reconstituted in DMSO at anominal concentration of 5 mg/mL glucagon. Formulations were placed instability incubators at 5° C., 25° C., and 40° C. Glucagon purity wasdetermined with a reverse phase HPLC method.

The stability of the formulation in glycine buffer was significantlygreater after 1 month of incubation at the various temperatures. Table 3below shows the RP-HPLC purity at various times of incubation at 40° C.

TABLE 3 Effect of buffer species on the stability of glucagon in DMSOFormulation Time = 0 1 week 2 weeks 4 weeks Glycine, pH 3.0 99.4 99.199.0 96.6 Citrate, pH 3.0 98.6 97.7 97.3 92.7

Example 10 Effect of Moisture on Glucagon Stability in DMSO

Glucagon acetate was prepared at 1.0 mg/mL via dissolution in one of thefollowing buffers:

1. 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl)

2. 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl)

These formulations were lyophilized and reconstituted in DMSO at anominal concentration of 5 mg/mL glucagon. Additional moisture was addedto the second formulation. Moisture content was measured using the KarlFisher method. The first formulation had a moisture content of 0.13%(w/w), whereas the second formulation had a moisture content of 0.54%(w/w). Formulations were placed in stability incubators at 5° C., 25°C., and 40° C. Glucagon purity was determined with a reverse phase HPLCmethod.

The stability of the formulation with lower moisture was significantlygreater after 1 month of incubation at the various temperatures. Table 4below shows the RP-HPLC purity at various times of incubation at 40° C.Even at moisture contents below 1%, a significant difference instability can be detected.

TABLE 4 Effect of residual moisture on the stability of glucagon in DMSOFormulation Time = 0 1 week 2 weeks 4 weeks Lower moisture 99.4 99.199.0 96.6 Additional moisture 99.2 98.9 98.8 95.6

Example 11 Freezing Point Depression of DMSO Solutions

Using PerkinElmer Instruments PYRIS Diamond Differential Scanningcalorimetry (“DSC”), samples were cooled to −40° C. and heated to 40° C.at 8° C. per minute for screening purposes.

DMSO/NMP Blends

Various DMSO and NMP blends were tested, including:

1. 90% DMSO+10% NMP

2. 80% DMSO+20% NMP

3. 70% DMSO+30% NMP

4. 60% DMSO+40% NMP

5. 50% DMSO+50% NMP

DSC scans showed that the temperature of crystallization of the solventsprogressively reduced from ˜18° C. for neat DMSO to −5.7° C. for a 50%NMP/50% DMSO blend. Addition of the glucagon acetate, glycine lyophileto a glucagon concentration of 5 mg/mL resulted in an additional ˜4° C.reduction in the freezing point.

DMSO/Ethyl Acetate Blends

Various DMSO and ethyl acetate blends were tested, including:

1. 80% DMSO+20% ethyl acetate (T_(c)=16° C.)

2. 70% DMSO+30% ethyl acetate

3. 60% DMSO+40% ethyl acetate (T_(c)=6.5° C.)

4. 50% DMSO+50% ethyl acetate (T_(c)=2.9° C.)

5. 40% DMSO+60% ethyl acetate (T_(c)=none observed)

DSC scans showed that the temperature of crystallization of the solventsprogressively reduced from ˜18° C. for neat DMSO to 2.9° C. for a 50%NMP/50% DMSO blend. No crystallization peak was observed for a 40%DMSO/60% ethyl acetate blend. Additionally, these formulations werestored at refrigerated temperature (4° C.) for several days and observedvisually for evidence of freezing. All formulations with 30% ethylacetate or greater in the co-solvent stayed liquid and did not freeze.This is somewhat different from the T_(c) observed in the DSC studies.

DMSO Solutions with Alcohol Co-Solvents

Various DMSO solutions to which an alcohol co-solvent (ethanol,glycerol, or propylene glycol) were added were tested, including:

1. 95% DMSO+5% alcohol

2. 90% DMSO+10% alcohol

3. 80% DMSO+20% alcohol

4. 70% DMSO+30% alcohol

5. 60% DMSO+40% alcohol

6. 50% DMSO+50% alcohol

7. 40% DMSO+60% alcohol

8. 30% DMSO+70% alcohol

9. 20% DMSO+80% alcohol

10. 10% DMSO+90% alcohol

These formulations were stored at refrigerated temperature (4° C.) forseveral days and observed visually for evidence of freezing. Allformulations with 20% alcohol co-solvent or greater stayed liquid anddid not freeze. DSC scans showed the freezing point of 20% alcoholco-solvents to be 2.3° C., 0.6° C., and 3.3° C. for ethanol, glycerol,and propylene glycol, respectively.

Example 12 Freeze-Thaw Stability of Glucagon

Glucagon acetate was prepared at 1.0 mg/mL via dissolution in 2 mML-glycine, pH 3.0 (titrated with concentrated HCl). The glucagonformulations were lyophilized and reconstituted in DMSO at a nominalconcentration of 5 mg/mL glucagon. Solution samples were divided andtrehalose was added to one solution to a concentration of 5%. Theseformulations were aliquoted into vials and placed in a stabilityincubator at 5° C. At 5° C., these solutions were observed to freeze.The glucagon solutions were thawed at various intervals and turbiditywas determined using the absorbance at 630 nm.

Table 5 below shows the turbidity of the glucagon solutions at varioustimes of incubation at 5° C. The solutions without trehalose showedincreases in turbidity at various timepoints of incubation. Solutionscontaining trehalose, however, showed no increase in turbidity. Theturbidity measurements were confirmed through visual observation.Samples frozen and incubated without trehalose were cloudy or hazy uponobservation.

TABLE 5 Turbidity of glucagon solutions after incubation at 5° C.Formulation Time = 0 1 week 2 weeks 4 weeks No trehalose 0.024 0.1420.130 0.160 5% trehalose 0.016 0.029 0.028 0.035

Surprisingly, use of a carbohydrate additive such as trehalose insolutions of peptides in DMSO enhances the stability of the peptideduring the freeze-thawing process.

Example 13 Enhanced Thawing Rate with Trehalose

Glucagon acetate was prepared at 1.0 mg/mL via dissolution in 2 mML-glycine, pH 3.0 (titrated with concentrated HCl) as described abovefor Example 13. Upon removal from storage at 5° C., samples of glucagonsolutions containing trehalose were observed to thaw completely in amuch shorter time than solutions without trehalose. Trehalose-containingsamples were observed to thaw completely in less than 30 seconds, ascontrasted with glucagon solutions without trehalose, which weretypically observed to thaw completely over several minutes. The abilityto quickly thaw a peptide formulation can be particularly advantageousin an emergency medical setting, in the event a solution was frozen andhad to be injected rapidly.

Example 14 Effect of pH on Insulin Solubility

Insulin was dissolved in H₂O at 10 mg/mL with a 10 mMphosphate/citrate-1 mM EDTA buffer at either pH 2 or pH 7. Thesesolutions were lyophilized to dryness (>1% residual moisture) using aconservative cycle and reconstituted to various nominal concentrationsin DMSO. Solubility was measured by visual inspection for clarity andturbidity via A₆₃₀.

At a pH of 2, insulin was observed to be soluble to concentrations of atleast 100 mg/mL. However, at a pH memory of 7, even at the lowestconcentration tested, 10 mg/mL, poor solubility of insulin was observedas cloudy or hazy solutions with increased light scattering (A₆₃₀). Somelower-concentration, e.g., 10 mg/mL, insulin solutions with a pH memoryof 7 were observed to slowly dissolve to a clear solution over a periodof about 24 hours.

Example 15 Effect of pH on Pramlintide Solubility

Pramlintide acetate was dissolved in H₂O at 2 mg/mL with either a 10 mMcitrate buffer, pH 4 or 10 mM phosphate buffer, pH 7. These solutionswere lyophilized to dryness (>1% residual moisture) using a conservativecycle and reconstituted to various nominal concentrations in DMSO.Solubility was measured by visual inspection for clarity and turbidityvia A₆₃₀.

At no concentration was pramlintide with a pH memory of 7 soluble inDMSO. However, a low concentration of pramlintide with a pH memory of 4was soluble in DMSO.

Example 16 Co-Formulations of Peptides in Aprotic Polar Solvents

Preparation of co-formulations are prepared by separately dryingformulations of the individual compounds from an aqueous solution thatprovides the optimal solubility/stability upon reconstitution into theaprotic polar solvent. Solution pH is a property that affects peptidesolubility, and a dried peptide, when reconstituted into an aproticpolar solvent, will retain a “pH memory” of the aqueous formulation fromwhich it was dried when a non-volatile buffer is used. Since aproticpolar solvents do not have exchangeable protons, the individual peptideswill maintain the solubility and stability characteristics of theoptimal pH memory.

Current pramlintide and insulin formulations conflict in their bufferingsystems, making compatibility of a mixed formulation difficult. Mostinsulins and insulin analogs have an isoelectric point in the range of5-6 and are thus formulated at a pH of around 7 or at a lower pH ofaround 2. Pramlintide has an isoelectric point of >10.5 and isformulated at a pH of around 4 where it is optimally stable. Theinteraction of pramlintide and insulin formulations at different pHs anddiffering buffering capacities often results in precipication of solubleinsulin components or solubilization of crystalline insulin components.In vitro studies with pramlintide and short- and long-acting insulinformulations found substantial variability in insulin solubility whenvarious quantities of insulin were mixed with fixed quantities ofpramlintide.

Thus, the present invention provides a formulation whereby both arapid-acting insulin species and an amylin analog are stable and can beadministered simultaneously from a single formulation for injection orformulation. This formulation more closely mimics the naturalphysiological response to post-prandial rise in blood glucose than theprior art.

Examples of peptides that can be co-formulated include, but are notlimited to: (1) insulin-amylin (insulin at a pH memory of about 2.0 orabout 7.0, and amylin or an amylin analog (e.g., pramlintide) at a pHmemory of about 4.0); and (2) glucagon-GLP-1 (glucagon at a pH memory ofabout 3.0 or below, and glucagon-like peptide-1 (GLP-1) or an analogthereof (e.g., exenatide) at a pH memory of about 4.0-5.0).

A co-formulation of insulin and pramlintide was prepared as follows: Aninsulin formulation of 100 mg/mL insulin, pH memory 2, was made asdescribed above in Example 14. A pramlintide formulation of 1 mg/mLpramlintide, pH memory 4, was made as described above in Example 15. 5μl of the insulin formulation was mixed with 95 ml of the pramlintidesolution. The resulting solution was observed to be clear and thuscreated a soluble co-formulation of insulin and pramlintide withrespective pH memory of 2 and 4, respectively.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents and PCT publicationsare incorporated herein by reference for all purposes.

1-98. (canceled)
 99. A method of treating hypoglycemia comprisingadministering a stable solution to a subject in need thereof, the stablesolution comprising: (a) a glucagon peptide or a salt thereof that hasbeen dried from a buffer, wherein the glucagon peptide or salt thereofhas a pH memory; and (b) an aprotic polar solvent, wherein the glucagonpeptide or salt thereof is reconstituted and solubilized in the aproticpolar solvent in an amount from about 0.1 mg/mL up to the solubilitylimit of the glucagon peptide or salt thereof, and wherein the glucagonpeptide or salt thereof maintains the pH memory in the aprotic polarsolvent.
 100. The method of claim 99, wherein the stable solution isadministered with a syringe.
 101. The method of claim 99, wherein thestable solution is administered with a pen injection device.
 102. Themethod of claim 99, wherein the stable solution is administered with anauto-injector device.
 103. The method of claim 99, wherein the stablesolution is administered with a pump device.
 104. The method of claim99, wherein the buffer is a non-volatile buffer.
 105. The method ofclaim 104, wherein the non-volatile buffer is selected from a glycinebuffer, a citrate buffer, a phosphate buffer, or mixtures thereof. 106.The method of claim 105, wherein the non-volatile buffer is a glycinebuffer.
 107. The method of claim 105, wherein the pH memory is aboutequal to the pH of the glucagon or salt thereof in the non-volatilebuffer.
 108. The method of claim 99, wherein the pH memory of theglucagon or salt thereof is from about 2 to
 3. 109. The method of claim99, wherein the pH memory of the glucagon or salt thereof is from 1 to4.
 110. The method of claim 99, wherein the aprotic polar solvent isselected from dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP),ethyl acetate, and mixtures thereof.
 111. The method of claim 110,wherein the aprotic polar solvent is n-methyl pyrrolidone (NMP). 112.The method of claim 111, wherein the aprotic polar solvent isdimethylsulfoxide (DMSO).
 113. The method of claim 99, wherein thestable solution further comprises a co-solvent that depresses thefreezing point of the formulation, wherein the co-solvent is selectedfrom ethanol, propylene glycol, glycerol, and mixtures thereof.
 114. Themethod of claim 99, wherein the stable solution further comprises astabilizing excipient selected from a sugar, a starch, and mixturesthereof.
 115. The method of claim 99, wherein the stable solutioncomprises from about 0.1 mg/mL to about 30 mg/mL of the glucagon or saltthereof.
 116. The method of claim 99, wherein the buffer is a glycinebuffer and the aprotic solvent is dimethylsulfoxide (DMSO).
 117. Themethod of claim 116, wherein the stable solution further comprises astabilizing excipient selected from sugars, starches, and mixturesthereof.
 118. The method of claim 117, wherein the stabilizing excipientis trehalose.
 119. The method of claim 99, wherein the water content ofthe stable solution is less than 5%.
 120. The method of claim 99,wherein the stable solution further comprises a second peptide or saltthereof.
 121. The method of claim 120, wherein the second peptide is aglucagon-like peptide-1 (GLP-1).
 122. The method of claim 99, whereinthe subject has hypoglycemia.
 123. The method of claim 99, wherein thestable solution is administered parenterally.
 124. The method of claim99, wherein the stable solution is administered into an intradermalregion of the subject.
 125. The method of claim 99, wherein the stablesolution is administered into a subcutaneous region of the subject. 126.The method of claim 99, wherein the stable solution is administered intoan intramuscular region of the subject.
 127. The method of claim 99,wherein the subject is a human.